Alpha-amylase variants

ABSTRACT

The present invention relates to alpha-amylase variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/EP2015/063135 filed Jun. 12, 2015, which claims priority or thebenefit under 35 U.S.C. 119 of U.S. provisional application No.62/011,564 filed Jun. 12, 2014, the contents of which are fullyincorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to alpha-amylase variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants.

Description of the Related Art

Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1)constitute a group of enzymes, which catalyses hydrolysis of starch andother linear and branched 1,4-gluosidic oligo- and polysaccharides.

Alpha-amylase is a key enzyme for use in detergent compositions and itsuse has become increasingly important for removal of starchy stainsduring laundry washing or dishwashing.

Some detergents, in particular dishwashing detergents, contain bleachingsystems, bleach activators, and bleach catalysts which are all verydestabilizing for the alpha-amylases due to oxidation of the molecules.Therefore, it is important to find alpha-amylase variants, which arestable, have high wash performance, stain removal effect and/or activityin detergents comprising various bleaching agents.

It is known in the art to stabilize alpha-amylases towards bleachingagents and oxidation by these by substituting the methionine at position197 (using the amylase from B. licheniformis for numbering) with e.g.leucine. This has e.g. been disclosed in WO199418314. However, theseprior art oxidation stable alpha-amylases have the disadvantage that thealpha-amylase activity is reduced.

Thus, it is an object of the present invention to provide alpha-amylasevariants that exhibit a high level of stability in detergents, inparticular in dishwashing detergents and other detergents comprisingbleaching agents or systems but at the same time have improvedalpha-amylase activity compared to the parent alpha-amylase having theprior art solution of M197L substitution. It is a further object toprovide alpha-amylase variants which have high performance, inparticular high wash performance, in particular high dishwashingperformance.

The present invention provides alpha-amylase variants with improvedstability compared to its parent and improved activity compared to itsparent having M197L (which in the parent amylases of the presentinvention corresponds to M202L).

SUMMARY OF THE INVENTION

The present invention relates to an isolated alpha-amylase variantcomprising a) a deletion at two or more positions corresponding topositions R181, G182, D183 and G184 of the mature polypeptide of SEQ IDNO: 1, and b) a substitution at one or more positions corresponding topositions Y198, Y200, L201, Y203 and A204 of the mature polypeptide ofSEQ ID NO: 1, and c) a substitution of the methionine at the positioncorresponding to position M202 of the mature polypeptide of SEQ ID NO: 1with any other amino acid, wherein the variant has at least 80%, such asat least 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 1 or 2, and wherein the variant hasalpha-amylase activity.

The present invention also relates to detergent compositions comprisingthe variants, isolated polynucleotides encoding the variants; nucleicacid constructs, vectors, and host cells comprising the polynucleotides;and methods of producing the variants.

The present invention also relates to the use of the variants in acleaning process.

The present invention also relates to a method of improving the activityof an alpha-amylase by introducing into a parent alpha-amylase a) adeletion at two or more positions corresponding to positions R181, G182,D183 and G184 of the mature polypeptide of SEQ ID NO: 1, and b) asubstitution at one or more positions corresponding to positions Y198,Y200, L201, Y203 and A204 of the mature polypeptide of SEQ ID NO: 1, andc) a substitution of the methionine at the position corresponding toposition M202 of the mature polypeptide of SEQ ID NO: 1 with any otheramino acid, wherein the resulting variant has at least 80%, such as atleast 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 1 or 2.

Definitions

Alpha-amylase: The term “alpha-amylase” as used herein, refers to anenzyme capable of catalyzing the degradation of starch. Generally,alpha-amylases (E.C. 3.2.1.1, α-D-(1→4)-glucan glucanohydrolase) areendo-acting enzymes that cleave the α-D(1→4) O-glycosidic linkageswithin the starch molecule in a random order.

The term “starch” as used herein, refers to any material comprised ofthe complex polysaccharide carbohydrates of plants, comprised of amyloseand amylopectin with the formula (C₆H₁₀O₅)_(x), wherein X can be anynumber. In particular, the term refers to plant-based materials, such asrice, barley, wheat, corn, rye, potato, and the like.

Alpha-amylase activity: The term “alpha-amylase activity” means theactivity of alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1, whichconstitute a group of enzymes, which catalyze hydrolysis of starch andother linear and branched 1,4-glucosidic oligo- and polysaccharides. Forpurposes of the present invention, alpha-amylase activity is determinedaccording to the procedure described in the Methods. In one aspect, thevariants of the present invention have at least 20%, e.g., at least 40%,at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, or at least 100% of the alpha-amylase activity of the maturepolypeptide of SEQ ID NO: 1 or 2.

SEQ ID NO: 1: HHDGTNGTIM QYFEWNVPND GQHWNRLHNN AQNLKNAGITAIWIPPAWKG TSQNDVGYGA YDLYDLGEFN QKGTVRTKYGTKAELERAIR SLKANGIQVY GDVVMNHKGG ADFTERVQAVEVNPQNRNQE VSGTYQIEAW TGFNFPGRGN QHSSFKWRWYHFDGTDWDQS RQLANRIYKF RGDGKAWDWE VDTENGNYDYLMYADVDMDH PEVINELNRW GVWYANTLNL DGFRLDAVKHIKFSFMRDWL GHVRGQTGKN LFAVAEYWKN DLGALENYLSKTNWTMSAFD VPLHYNLYQA SNSSGNYDMR NLLNGTLVQRHPSHAVTFVD NHDTQPGEAL ESFVQGWFKP LAYATILTREQGYPQVFYGD YYGIPSDGVP SYRQQIDPLL KARQQYAYGRQHDYFDHWDV IGWTREGNAS HPNSGLATIM SDGPGGSKWMYVGRQKAGEV WHDMTGNRSG TVTINQDGWG HFFVNGGSVS VWVKR SEQ ID NO: 2:HHDGTNGTIM QYFEWNVPND GQHWNRLHNN AQNLKNAGITAIWIPPAWKG TSQNDVGYGA YDLYDLGEFN QKGTVRTKYGTKAELERAIR SLKANGIQVY GDVVMNHKGG ADFTERVQAVEVNPQNRNQE VSGTYQIEAW TGFNFPGRGN QHSSFKWRWYHFDGTDWDQS RQLANRIYKF RG KAWDWE VDTEFGNYDYLMYADVDMDH PEVINELNRW GVWYANTLNL DGFRLDAVKHIKFSFMRDWL GHVRGQTGKN LFAVAEYWKN DLGALENYLSKTNWTMSAFD VPLHYNLYQA SNSSGNYDMR NLLNGTLVQRHPSHAVTFVD NHDTQPGEAL ESFVQGWFKP LAYATILTREQGYPQVFYGD YYGIPSDGVP SYRQQIDPLL KARQQYAYGRQHDYFDHWDV IGWTREGNAS HPNSGLATIM SDGPGGSKWMYVGRQKAGEV WHDMTGNRSG TVTINQDGWG HFFVNGGSVS VWVKR

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Corresponding to: The term “corresponding to” as used herein, refers toway of determining the specific amino acid of a sequence whereinreference is made to a specific amino acid sequence. E.g. for thepurposes of the present invention, when references are made to specificamino acid positions, the skilled person would be able to align anotheramino acid sequence to said amino acid sequence that reference has beenmade to, in order to determine which specific amino acid may be ofinterest in said another amino acid sequence. Alignment of another aminoacid sequence with e.g. the sequence as set forth in SEQ ID NO: 1, orany other sequence listed herein, has been described elsewhere herein.Alternative alignment methods may be used, and are well-known for theskilled person.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Detergent composition: The term “detergent composition” as used herein,refers to a composition suitable for use within the field of detergents,such as for use in laundry and dish wash. A detergent composition may bein the form of a liquid or powder form, and may be suitable for bothhandwash or automated wash. Thus, the term “detergent composition”includes otherwise indicated by context, granular or powder-formall-purpose or heavy-duty washing agents, especially the so-calledheavy-duty liquid (HDL) types; liquid fine-fabric detergents; handdishwashing agents or light duty dishwashing agents, especially those ofthe high-foaming type; machine dishwashing agents, including the varioustablet, granular, liquid and rinse-aid types for household andinstitutional use; liquid cleaning and disinfecting agents, includingantibacterial hand-wash types, cleaning bars, soap bars, mouthwashes,denture cleaners, car or carpet shampoos, bathroom cleaners; hairshampoos and hair-rinses; shower gels, foam baths; metal cleaners; aswell as cleaning auxiliaries such as bleach additives and “stain-stick”or pre-treat types. The terms “detergent composition” and “detergentformulation” are used in reference to mixtures which are intended foruse in a wash medium for the cleaning of soiled objects. In someembodiments, the term is used in reference to laundering fabrics and/orgarments (e.g., “laundry detergents”). In alternative embodiments, theterm refers to other detergents, such as those used to clean dishes,cutlery, etc. (e.g., “dishwashing detergents”). It is not intended thatthe present invention be limited to any particular detergent formulationor composition. The term “detergent composition” is not intended to belimited to compositions that contain surfactants. It is intended that inaddition to the variants herein described, the detergents compositionsmay comprise, e.g., surfactants, builders, chelators or chelatingagents, bleach system or bleach components, polymers, fabricconditioners, foam boosters, suds suppressors, dyes, perfume, tannishinhibitors, optical brighteners, bactericides, fungicides, soilsuspending agents, anti corrosion agents, enzyme inhibitors orstabilizers, enzyme activators, transferase(s), hydrolytic enzymes,oxido reductases, bluing agents and fluorescent dyes, antioxidants,and/or solubilizers.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has alpha-amylaseactivity. In one aspect, a fragment contains at least 480 amino acidresidues, at least 481 amino acid residues, or at least 482 amino acidresidues.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent orcompared to the mature polypeptide of SEQ ID NO: 1 or 2. Such improvedproperties include, but are not limited to, catalytic efficiency,catalytic rate, chemical stability, oxidation stability, pH activity, pHstability, specific activity, stability under storage conditions,substrate binding, substrate cleavage, substrate specificity, substratestability, surface properties, thermal activity, and thermo stability,and improved wash performance. Another property that may be improved inthe variants is the stability in detergent compositions, i.e. detergentstability. The detergent stability (or residual activity) of a givenvariant may be determined by incubating the variant in a detergent modelsolution preferably containing chelating agents such as EDTA, EGTA,DTPA, DTMPA, MGDA, EDDS, or HEDP.

Preferably, the variants of the present invention have improvedalpha-amylase activity compared to prior art oxidation stablealpha-amylases.

Wash performance: In the present context the term “wash performance” isused as an enzyme's ability to remove starch or starch-containing stainspresent on the object to be cleaned during e.g. laundry or hard surfacecleaning, such as dish wash. The wash performance may be quantified bycalculating the so-called delta remission value (ΔRem) as described inthe definition herein.

Improved wash performance: The term “improved wash performance” isdefined herein as a variant enzyme displaying an alteration of the washperformance of an amylase variant relative to the wash performance ofthe parent amylase or relative to the alpha-amylases of the prior arte.g. by increased stain removal. The term “wash performance” includescleaning in general e.g. hard surface cleaning as in dish wash, but alsowash performance on textiles such as laundry, and also industrial andinstitutional cleaning. Improved wash performance may be measured bycomparing of the so-called delta remission value (ΔRem) as described inthe definition herein, wherein the ΔRem of the variant is compared withthe ΔRem of the parent alpha-amylase.

Low temperature: “Low temperature” is a temperature of 5-35° C.,preferably 5-30° C., more preferably 5-25° C., more preferably 5-20° C.,most preferably 5-15° C., and in particular 5-10° C. In a preferredembodiment, “Low temperature” is a temperature of 10-35° C., preferably10-30° C., more preferably 10-25° C., most preferably 10-20° C., and inparticular 10-15° C.

Delta remission value (ΔRem): The terms “Delta remission” or “Deltaremission value” are defined herein as the result of a reflectance orremission measurement at 460 nm of a test material, e.g. a swatch CS-28(Center For Testmaterials BV, P.O. Box 120, 3133 KT Vlaardingen, theNetherlands) or a hard surface. The swatch is measured with at least oneother swatch, washed under identical conditions, as background. Thedelta remission is the remission value of the test material washed withamylase subtracted the remission value of the test material washedwithout amylase.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Immunological cross reactivity: The term “a polypeptide havingimmunological cross reactivity with an antibody” as used herein, refersto any polypeptide which is bound by an antibody raised against thepolypeptide of SEQ ID NO: 1 or 2. When a polypeptide not necessarilyhaving the sequence as set forth in SEQ ID NO: 1 or 2 is bound by anantibody, and thereby provides cross reactivity, it indicates that thepolypeptide may have similar characteristics as the polypeptides of SEQID NO: 1 or 2. Determination of cross reactivity may be done by ELISAcomprising the steps of (i) adhering the polypeptide of interest to theELISA plate; (ii) adding the antibody raised against the polypeptide ofSEQ ID NO: 1 or 2; (iii) adding a secondary labeled antibody binging theantibody raised against the polypeptide of SEQ ID NO: 1 or 2; and (iv)measuring the signal from the bound secondary antibody. Other methods ofdetermining the immunological cross reactivity may be used and is withinthe knowledge of the skilled person.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 485 of SEQ ID NO: 1.

It is known in the art that a host cell may produce a mixture of two ofmore different mature polypeptides (i.e., with a different C-terminaland/or N-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving alpha-amylase activity.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent alpha-amylase: The term “parent” or “parentalpha-amylase” means an alpha-amylase to which an alteration is made toproduce the enzyme variants of the present invention. The parent may bea naturally occurring (wild-type) polypeptide such e.g. as thealpha-amylase of SEQ ID NO: 1 or a variant or fragment thereof such ase.g. the amylase of SEQ ID NO: 2.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Variant: The term “variant” means a polypeptide having alpha-amylaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition. The variants of the present invention have at least 20%, e.g.,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 100% of the alpha-amylase activityof the mature polypeptide of SEQ ID NO: 1 or 2.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type alpha-amylase: The term “wild-type” alpha-amylase means analpha-amylase expressed by a naturally occurring microorganism, such asa bacterium, archaea, yeast, or filamentous fungus found in nature.

Fabric: The term “fabric” encompasses any textile material. Thus, it isintended that the term encompass garments, as well as fabrics, yarns,fibers, non-woven materials, natural materials, synthetic materials, andany other textile material.

Textile: The term “textile” refers to woven fabrics, as well as staplefibers and filaments suitable for conversion to or use as yarns, woven,knit, and non-woven fabrics. The term encompasses yarns made fromnatural, as well as synthetic (e.g., manufactured) fibers. The term,“textile materials” is a general term for fibers, yarn intermediates,yarn, fabrics, and products made from fabrics (e.g., garments and otherarticles).

Non-fabric detergent compositions: The term “non-fabric detergentcompositions” include non-textile surface detergent compositions,including but not limited to compositions for hard surface cleaning,such as dishwashing detergent compositions, oral detergent compositions,denture detergent compositions, and personal cleansing compositions.

Effective amount of enzyme: The term “effective amount of enzyme” refersto the quantity of enzyme necessary to achieve the enzymatic activityrequired in the specific application, e.g., in a defined detergentcomposition. Such effective amounts are readily ascertained by one ofordinary skill in the art and are based on many factors, such as theparticular enzyme used, the cleaning application, the specificcomposition of the detergent composition, and whether a liquid or dry(e.g., granular, bar) composition is required, and the like. The term“effective amount” of a variant refers to the quantity of variantdescribed hereinbefore that achieves a desired level of enzymaticactivity, e.g., in a defined detergent composition. In one embodiment,the effective amount of a protease variant is the same effective amountof an alpha-amylase, such as an alpha-amylase variant. In anotherembodiment, the effective amount of a protease variant is different thanthe effective amount of an alpha-amylase, such as an alpha-amylasevariant, e.g., the effective amount of a protease variant may be more ormay be less than the effective amount of an alpha-amylase, such as analpha-amylase variant.

Water hardness: The term “water hardness” or “degree of hardness” or“dH” or “° dH” as used herein refers to German degrees of hardness. Onedegree is defined as 10 milligrams of calcium oxide per liter of water.

Relevant washing conditions: The term “relevant washing conditions” isused herein to indicate the conditions, particularly washingtemperature, time, washing mechanics, detergent concentration, type ofdetergent and water hardness, actually used in households in a detergentmarket segment.

Stain removing enzyme: The term “stain removing enzyme” as used herein,describes an enzyme that aids the removal of a stain or soil from afabric or a hard surface. Stain removing enzymes act on specificsubstrates, e.g., protease on protein, amylase on starch, lipase andcutinase on lipids (fats and oils), pectinase on pectin andhemicellulases on hemicellulose. Stains are often depositions of complexmixtures of different components which either results in a localdiscolouration of the material by itself or which leaves a stickysurface on the object which may attract soils dissolved in the washingliquor thereby resulting in discolouration of the stained area. When anenzyme acts on its specific substrate present in a stain the enzymedegrades or partially degrades its substrate thereby aiding the removalof soils and stain components associated with the substrate during thewashing process.

Reduced amount: The term “reduced amount” means in this context that theamount of the component is smaller than the amount which would be usedin a reference process under otherwise the same conditions. In apreferred embodiment the amount is reduced by, e.g., at least 5%, suchas at least 10%, at least 15%, at least 20% or as otherwise hereindescribed.

Low detergent concentration: The term “low detergent concentration”system includes detergents where less than about 800 ppm of detergentcomponents is present in the wash water. Asian, e.g., Japanesedetergents are typically considered low detergent concentration systems.

Medium detergent composition: The term “medium detergent concentration”system includes detergents wherein between about 800 ppm and about 2000ppm of detergent components is present in the wash water. North Americandetergents are generally considered to be medium detergent concentrationsystems.

High detergent concentration: The term “high detergent concentration”system includes detergents wherein greater than about 2000 ppm ofdetergent components is present in the wash water. European detergentsare generally considered to be high detergent concentration systems.

Liquid laundry detergent composition: The term “liquid laundry detergentcomposition” as used herein refers to a detergent composition which isin a stabilized liquid form and used in a method for laundering afabric. Thus, the detergent composition has been formulated to be influid form.

Powder laundry detergent composition: The term “powder laundry detergentcomposition” as used herein refers to a detergent composition which isin a solid form, such as a granulate, non-dusting granulate or powder,which is used in a method for laundering a fabric.

Liquid dishwash detergent composition: The term “liquid dishwashdetergent composition” as used herein refers to a detergent compositionwhich is in a stabilized liquid form and used in dishwash. Dishwash maybe any kind of dishwash, such as manual dishwash and such as automateddishwash (ADW).

Powder dishwash detergent composition: The term “powder dishwashdetergent composition” as used herein refers to a detergent compositionwhich is in a solid form, such as a granulate, powder or compact unitand used in dishwash. A powder dishwash detergent composition istypically used in automated dishwash, but the used is not limited tosuch ADW, and may also be intended for used in any other kind ofdishwash, such as manual dishwash.

Polynucleotide encoding: The term “polynucleotide encoding” as usedherein, refers to a polynucleotide that encodes a mature polypeptidehaving alpha-amylase activity.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide disclosedin SEQ ID NO: 1 is used to determine the corresponding amino acidresidue in another alpha-amylase. The amino acid sequence of anotheralpha-amylase is aligned with the mature polypeptide disclosed in SEQ IDNO: 1, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed in SEQ ID NO: 1 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotheralpha-amylase may be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010,Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 1 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms may be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitymay be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,may be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments may inturn be used to generate homology models for the polypeptide, and suchmodels may be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmsmay additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviations are employed.

Substitutions: For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. In situations where theamino acid at a given position may be substituted for any other aminoacid it is designated T226ACDEFGHIKLMNPQRSWVY. Accordingly, this meansthat threonine at position 226 may be substituted with one amino acidselected from the group of A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, W, V or Y. Likewise, in situations where the amino acid at a givenposition may be substituted for one amino acid selected from a specificgroup of amino acids, e.g. where the threonine at position 226 may besubstituted with any of tyrosine, phenylalanine or histidine it isdesignated T226YFH. The different alterations at a given position mayalso be separated by a comma (“,”), e.g., “Arg170Tyr,Glu” or “R170Y,E”represents a substitution of arginine at position 170 with tyrosine orglutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala” designates thefollowing variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”,“Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Multiple alterations are separated by addition marks (“+”), e.g.,“Gly205Arg+Ser411Phe” or “G205R+S411F”, representing substitutions atpositions 205 and 411 of glycine (G) with arginine (R) and serine (S)with phenylalanine (F), respectively.

Deletions: For an amino acid deletion, the following nomenclature isused: Original amino acid, position,*. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g.,“Glyl195*+Ser411*” or “G195*+S411*”.

Insertions: For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G-K-A

Multiple alterations: Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively. Theterm “alteration”, the term “mutation”, the term “variation”, and theterm “modification” may be used interchangeably and constitute the samemeaning and purpose unless otherwise stated by context.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated alpha-amylase variantcomprising a) a deletion at two or more positions corresponding topositions R181, G182, D183 and G184 of the mature polypeptide of SEQ IDNO: 1, and b) a substitution at one or more positions corresponding topositions Y198, Y200, L201, Y203, and A204 of the mature polypeptide ofSEQ ID NO: 1, and c) a substitution of the methionine at the positioncorresponding to position M202 of the mature polypeptide of SEQ ID NO: 1with any other amino acid, wherein the variant has at least 80%, such asat least 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 1 or 2, and wherein the variant hasalpha-amylase activity. In another embodiment the variant has at least80%, such as at least 90%, but less than 100% sequence identity with themature polypeptide of SEQ ID NO: 3. In another embodiment, the varianthas at least 80%, such as at least 90%, but less than 100% sequenceidentity with the mature polypeptide of SEQ ID NO: 4. In anotherembodiment, the variant has at least 80%, such as at least 90%, but lessthan 100% sequence identity with the mature polypeptide of SEQ ID NO: 5.In another embodiment, the variant has at least 80%, such as at least90%, but less than 100% sequence identity with the mature polypeptide ofSEQ ID NO: 6. In another embodiment, the variant has at least 80%, suchas at least 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 7. In another embodiment, the variant has atleast 80%, such as at least 90%, but less than 100% sequence identitywith the mature polypeptide of SEQ ID NO: 8.

The term “a substitution with any other amino acid” as used herein,refers to substituting the amino acid originally occurring at theparticular position with any of the nineteen alternativenaturally-occurring amino acids, i.e. when the originally occurringamino acid is an A, “any other amino acid” may be any one of thefollowing; R, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V.Any other amino acid is not limited to naturally-occurring amino acidsbut may also be non-naturally-occurring amino acids.

In one embodiment the alteration b) is an insertion in the loop spanningfrom amino acid positions 199-204. The insertion may be selected fromthe list comprising: D199DX, Y200YX, L201LX, M202MX, Y203YX, A204AX,where X denotes any amino acid.

In one embodiment the alteration b) is selected from one or more ofY198FLYHQ, Y200 FLSYCWPHQRIMTNKVADEG, L201 FLSYCWPHQRIMTNKVADEG,Y203FLSYCWPHQRIMTNKVADEG, A204IMTSRVAG and the alteration c) is any ofM202FLSYCWPHQRIMTNKVADEG.

In preferred embodiments, the invention relates to variants comprisingan amino acid sequence of positions 198-204 (using SEQ ID NO: 1 fornumbering) selected from the list consisting of: YDYLLFA, YDWLLYA,YDWLLFA, YDWLLPA, YDYLLIA, YDYLLNA, YDYLLPA, YDYQLYA, YDPLLYA, YDYLLTA,YDYLLWA, YDNLLYA, YDQLLLA, YDQLLYA, YDWLLWA, YDYLLVA, YDQLLPA, YDQLLWA,YDYLLHA, YDYLLLA, YDYLLSA, YDYPLYA, YDYQPAA, YDELLYA, YDKLLPA, YDQLLNA,YDYELYA, YDYHLYA, YDYLLDA, YDYLPRA, YDYQLLA, YDYQLPA, YDYQLQA, YDDLLYA,YDKLLYA, YDWLLHA, YDWLLTA, YDWLPSA, YDWQLYA, YDYGLYA, YDYLLEA, YDYLLQA,YDYPLPA, YDYRLYA, YDIELSA, YDQLLIA, YDQLLSA, YDWLGYA, YDWLLAA, YDWQLHA,YDWWLPA, YDYELLA, YDYLFTA, YDYLLKA, YDYLYSA, YDYQLFA, YDYQYYA, YDYTLYA,YDYYLYA, YDIELWA, YDIELYA, YDNLLNA, YDNLLPA, YDPLLHA, YDQLLVA, YDQLPYA,YDWLLRA, YDWLWYA, YDWWLGA, YDYHLIA, YDYQHYA, YDYQLGA, YDYQLIA, YDYWLPA,YDELLWA, YDHLLNA, YDIELLA, YDIELNA, YDIELRA, YDILLYA, YDKLLWA, YDLPLYA,YDNLLLA, YDPLLAA, YDPLLPA, YDQHLPA, YDQLLEA, YDQLLQA, YDQLNYA, YDQLPFA,YDQLPNA, YDQLPRA, YDTLLLA, YDTLLYA, YDVLLYA, YDWLLKA, YDWLLLA, YDWLLNA,YDWLLQA, YDWLLVA, YDWLPPA, YDWLPTA, YDWPWYA, YDWWLWA, YDYHLFA, YDYHPSA,YDYLLNT, YDYLLYT, YDYLPFA, YDYLPSA, YDYLVSA, YDYLYPA, YDYLYRA, YDYPLFA,YDYPLSA, YDYPLTA, YDYPQYA, YDYQLTA, YDYQLWA, YDYQNYA, YDYQPRA, YDYQSHA,YDYREYA, YDYRLPA, YDYRNSA, YDYRPRA, YDYTQYA, YDYVLYA, YDYWLSA, YDDLLSA,YDELLDA, YDELLPA, YDELLTA, YDEQLEA, YDEQLYA, YDGLPHA, YDIELFA, YDIELKA,YDIELPA, YDKLLNA, YDKPPSA, YDLLLFA, YDLPLLA, YDNLLKA, YDPLKFA, YDPLLEA,YDPLLFA, YDPLLKA, YDPLLWA, YDPPLPA, YDPPLYA, YDPTLPA, YDPTQYA, YDQELPA,YDQLDHA, YDQLEYA, YDQLLDA, YDQLLFA, YDQLLNT, YDQLLYS, YDQLLYT, YDQLPSA,YDQLWYA, YDQQLVA, YDTLLWA, YDTPLFA, YDTPLYA, YDWELYA, YDWHLYA, YDWLHSA,YDWLLEA, YDWLLIA, YDWLLSA, YDWLNYA, YDWLPFA, YDWLPRA, YDWLQPA, YDWLQYA,YDWLWGA, YDWPLHA, YDWQLRA, YDWQLTA, YDWSLPA, YDWSLYA, YDWWLYA, YDYELEA,YDYELNA, YDYGLAA, YDYHEWA, YDYHLPA, YDYHLSA, YDYHQYA, YDYHTSA, YDYLFQA,YDYLHLA, YDYLIEA, YDYLLFT, YDYLLRA, YDYLLYG, YDYLPDA, YDYLPQA, YDYLPWA,YDYLQEA, YDYPGYA, YDYPHSA, YDYPLLA, YDYPLNA, YDYPNYA, YDYPSRA, YDYPWYA,YDYQEYA, YDYQLAA, YDYQLKA, YDYQLPT, YDYQPTA, YDYQPYA, YDYQQYA, YDYQWYA,YDYRLFA, YDYRPSA, YDYRTFA, YDYRTSA, YDYRTYA, YDYSLYA, YDYSVYA, YDYTPRA,YDYWLFA, YDYWLGA, YDYWLWA, YDYWLYA, YDDLLLA, YDDLLNA, YDDLPFA, YDEHLHA,YDELLFA, YDELLSA, YDELQIA, YDEWPYA, YDGLLSA, YDHLLYA, YDIELHA, YDIELKT,YDIELTA, YDIEVSA, YDIPLYA, YDIRGYA, YDIRNYA, YDIRTKA, YDIWLYA, YDKLPHA,YDKLQYA, YDKPLSA, YDLLLVA, YDNHLPA, YDNHLYA, YDNLGYA, YDNLLIA, YDNLLVA,YDNLLWA, YDNLPRA, YDNQLYA, YDNRLYA, YDPHRHA, YDPLHVA, YDPLLDA, YDPLLLA,YDPLQYA, YDPPQFA, YDPQLIA, YDQELYA, YDQLFSA, YDQLKYA, YDQLLAA, YDQLLHA,YDQLNNA, YDQLPAA, YDQLPPA, YDQLQNA, YDQLWGT, YDQLWPA, YDQLYPA, YDQNLYA,YDQPLPA, YDQQLQA, YDQTLYA, YDQWLHA, YDQWLTA, YDRLLPA, YDSELYA, YDTLIRA,YDTLLKA, YDTLLNA, YDTPLNA, YDTPLPA, YDTPQIA, YDTRLYA, YDTSLPA, YDTTLPA,YDTWKYA, YDVLLPA, YDVLNTA, YDWELIA, YDWHLPA, YDWHQYA, YDWHSHA, YDWHTQA,YDWLHHA, YDWLHYA, YDWLNWA, YDWLPAA, YDWLPGA, YDWLPIA, YDWLQVA, YDWLTPA,YDWLTQA, YDWNLSA, YDWNWYA, YDWPGYA, YDWPLIA, YDWPLVA, YDWPLYA, YDWPTYA,YDWQLIA, YDWQLLA, YDWQLNA, YDWWLDA, YDYDLYA, YDYEKYA, YDYELIA, YDYELPA,YDYELTA, YDYELWA, YDYGWPA, YDYGWYA, YDYHENA, YDYHHEA, YDYHIEA, YDYHLQA,YDYHPRA, YDYHTIA, YDYHTPA, YDYHTYA, YDYLFPA, YDYLHWA, YDYLIRA, YDYLNDA,YDYLNPA, YDYLNQA, YDYLPEA, YDYLPHA, YDYLPIA, YDYLPLA, YDYLPTA, YDYLQNA,YDYLQRA, YDYLRQA, YDYLWGA, YDYLWLA, YDYLWPA, YDYPEPA, YDYPIDA, YDYPIRA,YDYPLAA, YDYPLQA, YDYPPRA, YDYPQHA, YDYPSYA, YDYPTAA, YDYPTDA, YDYQHRA,YDYQHSA, YDYQIYA, YDYQLEA, YDYQLLT, YDYQNPA, YDYQQNA, YDYQQSA, YDYQTVA,YDYQWPA, YDYQYRA, YDYRHTA, YDYRLNA, YDYRQYA, YDYRRSA, YDYRSDA, YDYSGYA,YDYSNYA, YDYSTFA, YDYTEYA, YDYTLSA, YDYTQSA, YDYWLEA, YDYWLGT, YDYWLHA,YDYWLLA, YDYWLTA, YDYWPEA, YDYWPRA, YDYYLRA.

In a preferred embodiment the alteration b) and/or c) is selected fromone or more of Y200H, Y200Q, M202L, Y203N, Y203L, A204S, Y200H+M202L,Y200Q+M202L, M202L+Y203N, M202L+Y203L, M202L+A204S, Y200H+M202L+Y203N,Y200H+M202L+Y203L, Y200H+M202L+A204S, Y200Q+M202L+Y203N,Y200Q+M202L+Y203L, Y200Q+M202L+A204S, Y200H+M202L+Y203N+A204S,Y200H+M202L+Y203L+A204S, Y200Q+M202L+Y203N+A204S,Y200Q+M202L+Y203L+A204S.

In another preferred embodiment, the alteration b) and/or c) comprisesor consists of a substitution selected from the list consisting ofY200H, Y200Q, M202L, Y203N, Y203L, A204S or an insertion selected fromM202MG or M202GG.

In a preferred embodiment, the deletion a) is selected from the listconsisting of R181*+G182*, R181*+D183*, R181*+G184*, G182*+D183*,G182*+G184* and D183*+G184*, preferably D183*+G184*.

In a preferred embodiment, the deletion a) and alterations b) and c) areselected from the list consisting of: R181*+G182*+Y200H+M202L,R181*+G182*+Y200Q+M202L, R181*+G182*+M202L+Y203N,R181*+G182*+M202L+Y203L, R181*+G182*+M202L+A204S,R181*+G182*+Y200H+M202L+Y203N, R181*+G182*+Y200H+M202L+Y203L,R181*+G182*+Y200H+M202L+A204S, R181*+G182*+Y200Q+M202L+Y203N,R181*+G182*+Y200Q+M202L+Y203L, R181*+G182*+Y200Q+M202L+A204S,R181*+G182*+Y200H+M202L+Y203N+A204S,R181*+G182*+Y200H+M202L+Y203L+A204S,R181*+G182*+Y200Q+M202L+Y203N+A204S,R181*+G182*+Y200Q+M202L+Y203L+A204S, R181*+D183*+Y200H+M202L,R181*+D183*+Y200Q+M202L, R181*+D183*+M202L+Y203N,R181*+D183*+M202L+Y203L, R181*+D183*+M202L+A204S,R181*+D183*+Y200H+M202L+Y203N, R181*+D183*+Y200H+M202L+Y203L,R181*+D183*+Y200H+M202L+A204S, R181*+D183*+Y200Q+M202L+Y203N,R181*+D183*+Y200Q+M202L+Y203L, R181*+D183*+Y200Q+M202L+A204S,R181*+D183*+Y200H+M202L+Y203N+A204S,R181*+D183*+Y200H+M202L+Y203L+A204S,R181*+D183*+Y200Q+M202L+Y203N+A204S,R181*+D183*+Y200Q+M202L+Y203L+A204S, G182*+G184*+Y200H+M202L,G182*+G184*+Y200Q+M202L, G182*+G184*+M202L+Y203N,G182*+G184*+M202L+Y203L, G182*+G184*+M202L+A204S,G182*+G184*+Y200H+M202L+Y203N, G182*+G184*+Y200H+M202L+Y203L,G182*+G184*+Y200H+M202L+A204S, G182*+G184*+Y200Q+M202L+Y203N,G182*+G184*+Y200Q+M202L+Y203L, G182*+G184*+Y200Q+M202L+A204S,G182*+G184*+Y200H+M202L+Y203N+A204S,G182*+G184*+Y200H+M202L+Y203L+A204S,G182*+G184*+Y200Q+M202L+Y203N+A204S,G182*+G184*+Y200Q+M202L+Y203L+A204S, D183*+G184*+Y200H+M202L,D183*+G184*+Y200Q+M202L, D183*+G184*+M202L+Y203N,D183*+G184*+M202L+Y203L, D183*+G184*+M202L+A204S,D183*+G184*+Y200H+M202L+Y203N, D183*+G184*+Y200H+M202L+Y203L,D183*+G184*+Y200H+M202L+A204S, D183*+G184*+Y200Q+M202L+Y203N,D183*+G184*+Y200Q+M202L+Y203L, D183*+G184*+Y200Q+M202L+A204S,D183*+G184*+Y200H+M202L+Y203N+A204S,D183*+G184*+Y200H+M202L+Y203L+A204S,D183*+G184*+Y200Q+M202L+Y203N+A204S, andD183*+G184*+Y200Q+M202L+Y203L+A204S.

In another embodiment, the alteration a) and variation b) are selectedfrom the list consisting of N195F+M202L+R181*+G182*,N195F+M202L+R181*+D183*, N195F+M202L+R181*+G184*,N195F+M202L+G182*+D183*, N195F+M202L+D183*+G184*,N195Y+M202L+R181*+G182*, N195Y+M202L+R181*+D183*,N195Y+M202L+R181*+G184*, N195Y+M202L+G182*+D183* andN195Y+M202L+D183*+G184*.

In another embodiment, the alteration a) and alteration b) are selectedfrom the list consisting of N195F+M202L+R181*G182*,N195F+M202L+R181*+D183*, N195F+M202L+R181*+G184*,N195F+M202L+G182*+D183*, N195F+M202L+D183*+G184*,N195Y+M202L+R181*+G182*, N195Y+M202L+R181*+D183*,N195Y+M202L+R181*+G184*, N195Y+M202L+G182*+D183* andN195Y+M202L+D183*+G184*.

In another embodiment, the alteration a) and variation b) are selectedfrom the list consisting of R181*+G182*+N195F+Y200H+M202L+Y203N,R181*+G182*+N195F+Y200Q+M202L+A204S, R181*+G182*+N195F+M202L+Y203L,G182*+G184*+N195F+Y200H+M202L+Y203N,G182*+G184*+N195F+Y200Q+M202L+A204S, G182*+G184*+N195F+M202L+Y203L,G182*+D183*+N195F+Y200H+M202L+Y203N,G182*+D183*+N195F+Y200Q+M202L+A204S, G182*+D183*+N195F+M202L+Y203L,R181*+D184*+N195F+Y200H+M202L+Y203N,R181*+D184*+N195F+Y200Q+M202L+A204S, R181*+D184*+N195F+M202L+Y203L.

In another embodiment, the alteration a) and alteration b) are selectedfrom the list consisting of R181*+G182*+N195F+Y200H+M202L+Y203N,R181*+G182*+N195F+Y200Q+M202L+A204S, R181*+G182*+N195F+M202L+Y203L,G182*+G184*+N195F+Y200H+M202L+Y203N,G182*+G184*+N195F+Y200Q+M202L+A204S, G182*+G184*+N195F+M202L+Y203L,G182*+D183*+N195F+Y200H+M202L+Y203N,G182*+D183*+N195F+Y200Q+M202L+A204S, G182*+D183*+N195F+M202L+Y203L,R181*+D184*+N195F+Y200H+M202L+Y203N,R181*+D184*+N195F+Y200Q+M202L+A204S, R181*+D184*+N195F+M202L+Y203L.

In another embodiment, the alteration b) is at two or more of saidpositions, such as three or more of said positions, four or more of saidpositions, five or more of said positions, six or more of saidpositions, seven or more of said positions, eight or more of saidpositions, or nine or more of said positions.

In another embodiment, the number of alterations is 2-20, e.g., 2-10 and2-5, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity to the amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 3.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 4.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 5.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 6.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 7.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 8.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 9.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 10.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 11.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 12.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 13.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 14.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 15.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 16.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 17.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 18.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 19.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 20.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 21.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 22.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 23.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 24.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 25.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 26.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 27.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 28.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 29.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 30.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 31.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 32.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 33.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 34.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 35.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 36.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 37.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 38.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 39.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 40.

In another embodiment, the variant has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95% identity, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%,sequence identity, to the amino acid sequence of SEQ ID NO: 41.

In a preferred embodiment the variant is a variant of a parentalpha-amylase selected from the group consisting of: a. a polypeptidehaving at least 80% sequence identity to the mature polypeptide of SEQID NO: 1; b. a polypeptide having at least 80% sequence identity to themature polypeptide of SEQ ID NO: 2; c. a fragment of the maturepolypeptide of SEQ ID NO: 1, which has alpha-amylase activity; d. afragment of the mature polypeptide of SEQ ID NO: 2, which hasalpha-amylase activity; e. a polypeptide having immunological crossreactivity with an antibody raised against the mature polypeptide of SEQID NO: 1; f.

a polypeptide having immunological cross reactivity with an antibodyraised against the mature polypeptide of SEQ ID NO: 2.

In one embodiment, the parent alpha-amylase has at least 85%, such as atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 1.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 2.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 3.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 4.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 5.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 6.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 7.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 8.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 9.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 10.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 11.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 12.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 13.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 14.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 15.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 16.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 17.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 18.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 19.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 20.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 21.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 22.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 23.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 24.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 25.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 26.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 27.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 28.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 29.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 30.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 31.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 32.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 33.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 34.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 35.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 36.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 37.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 38.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 39.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 40.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to SEQ ID NO: 41.

In one embodiment, the parent alpha-amylase comprises or consists of themature polypeptide of SEQ ID NO: 1.

In another embodiment, the parent alpha-amylase comprises or consists ofthe mature polypeptide of SEQ ID NO: 2.

In a preferred embodiment, the variant has an improved property relativeto the parent alpha-amylase, wherein the improved property is selectedfrom the group consisting of catalytic efficiency, catalytic rate,chemical stability, oxidation stability, pH activity, pH stability,specific activity, stability under storage conditions, substratebinding, substrate cleavage, substrate specificity, substrate stability,surface properties, thermal activity, thermo stability, and preferablyimproved washing performance at low temperature.

In a particularly preferred embodiment, the variant has improveddetergent stability compared to the parent alpha-amylase.

In another embodiment, the variant has improved oxidation stability indetergents relative to the alpha-amylase of SEQ ID NO: 1 or 2.

In one embodiment, the variant comprises the deletion R181*+G182* of themature polypeptide of SEQ ID NO: 1.

In one embodiment, the variant comprises the deletion R181*+D183* of themature polypeptide of SEQ ID NO: 1.

In one embodiment, the variant comprises the deletion R181*+G184* of themature polypeptide of SEQ ID NO: 1.

In one embodiment, the variant comprises the deletion G182*+D183* of themature polypeptide of SEQ ID NO: 1.

In one embodiment, the variant comprises the deletion D183*+G184* of themature polypeptide of SEQ ID NO: 1.

In one embodiment, the variant has at least 80% sequence identity to theamino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 85% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 90% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 91% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 92% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 93% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 94% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 95% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 96% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 97% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 98% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the variant has at least 99% sequence identity tothe amino acid sequence of SEQ ID NO: 1.

In one embodiment, the variant has at least 80% sequence identity to theamino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 85% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 90% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 91% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 92% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 93% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 94% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 95% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 96% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 97% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 98% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

In another embodiment, the variant has at least 99% sequence identity tothe amino acid sequence of SEQ ID NO: 2.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum,improve wash performance, and the like.

Essential amino acids in a polypeptide may be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for alpha-amylase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction may also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidsmay also be inferred from an alignment with a related polypeptide.

In an embodiment, the variant has improved catalytic efficiency comparedto the alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved catalytic rate compared tothe alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved chemical stability comparedto the alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved oxidation stability comparedto the alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved detergent stability comparedto the alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved pH activity compared to thealpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved pH stability compared to thealpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved specific activity compared tothe alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved stability under storageconditions compared to the alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has decreased substrate binding comparedto the alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved substrate specificitycompared to the alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved substrate stability comparedto the alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved surface properties comparedto the alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved thermal activity compared tothe alpha-amylase of SEQ ID NO: 1 or 2.

In an embodiment, the variant has improved thermostability compared tothe alpha-amylase of SEQ ID NO: 1 or 2.

In another embodiment, the variant has improved wash performance, inparticular improved wash performance at low temperature compared to thealpha-amylase of SEQ ID NO: 1 or 2.

Parent Alpha-Amylases

The parent alpha-amylase may be (a) a polypeptide having at least 80%sequence identity to the mature polypeptide of SEQ ID NO: 1; (b) afragment of the mature polypeptide of SEQ ID NO: 1, which hasalpha-amylase activity; or (c) a polypeptide having immunological crossreactivity with an antibody raised against the mature polypeptide of SEQID NO: 1.

In another aspect, the parent alpha-amylase may be (a) a polypeptidehaving at least 80% sequence identity to the mature polypeptide of SEQID NO: 2; (b) a fragment of the mature polypeptide of SEQ ID NO: 2,which has alpha-amylase activity; or (c) a polypeptide havingimmunological cross reactivity with an antibody raised against themature polypeptide of SEQ ID NO: 2.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 1 of at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In one aspect, the aminoacid sequence of the parent differs by no more than 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQID NO: 1.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 of at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In one aspect, the aminoacid sequence of the parent differs by no more than 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQID NO: 2.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 3 of at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In one aspect, the aminoacid sequence of the parent differs by no more than 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQID NO: 3. In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 4 of at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In one aspect, the aminoacid sequence of the parent differs by no more than 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQID NO: 4 In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 5 of at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In one aspect, the aminoacid sequence of the parent differs by no more than 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQID NO: 5. In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 6 of at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In one aspect, the aminoacid sequence of the parent differs by no more than 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQID NO: 6. In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 7 of at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In one aspect, the aminoacid sequence of the parent differs by no more than 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQID NO: 7. In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 8 of at least 85%, at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%. In one aspect, the aminoacid sequence of the parent differs by no more than 10 amino acids,e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQID NO:8.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 9.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 10.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 11.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 12.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 13.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 14.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 15.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 16.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 17.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 18.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 19.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 20.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 21.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 22.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 23.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 24.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 25.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 26.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 27.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 28.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 29.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 30.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 31.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 32.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 33.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 34.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 35.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 36.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 37.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 38.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 39.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 40.

In another embodiment, the parent alpha-amylase has at least 85%, suchas at least 86%, at least 87%, at least 88%, at least 89%, at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%identity, at least 96%, at least 97%, at least 98%, or at least 99% or100% sequence identity to SEQ ID NO: 41.

In another aspect, the parent alpha-amylase comprises or consists of theamino acid sequence of SEQ ID NO: 1. In another aspect, the parentcomprises or consists of the amino acid sequence of SEQ ID NO: 2. Inanother aspect, the parent alpha-amylase comprises or consists of theamino acid sequence of SEQ ID NO: 3. In another aspect, the parentalpha-amylase comprises or consists of the amino acid sequence of SEQ IDNO: 4. In another aspect, the parent alpha-amylase comprises or consistsof the amino acid sequence of SEQ ID NO: 5. In another aspect, theparent alpha-amylase comprises or consists of the amino acid sequence ofSEQ ID NO: 6. In another aspect, the parent alpha-amylase comprises orconsists of the amino acid sequence of SEQ ID NO: 7. In another aspect,the parent alpha-amylase comprises or consists of the amino acidsequence of SEQ ID NO: 8.

In yet another embodiment, the parent alpha-amylase is an allelicvariant of the mature polypeptide of SEQ ID NO: 1 or 2.

The parent alpha-amylase may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide may further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent alpha-amylase may be obtained from microorganisms of anygenus. For purposes of the present invention, the term “obtained from”as used herein in connection with a given source shall mean that theparent encoded by a polynucleotide is produced by the source or by astrain in which the polynucleotide from the source has been inserted. Inone aspect, the parent alpha-amylase is secreted extracellularly.

The parent alpha-amylase may be a bacterial alpha-amylase. For example,the parent alpha-amylase may be a Gram-positive bacterial polypeptidesuch as a Bacillus alpha-amylase. In one aspect, the parentalpha-amylase is a Bacillus sp. AAI-10 alpha-amylase e.g., thealpha-amylase of SEQ ID NO: 1.

The alpha-amylases of SEQ ID NOs 1 and 2 as well as the variants hereofmay be artificially manufactured by methods known in the art.

Preparation of Variants

The present invention also relates to a method of improving the activityof an alpha-amylase by introducing into a parent alpha-amylase a) adeletion at two or more positions corresponding to positions R181, G182,D183 and G184 of the mature polypeptide of SEQ ID NO: 1, and b) asubstitution at one or more positions corresponding to positions Y198,Y200, L201, Y203 and A204 of the mature polypeptide of SEQ ID NO: 1, andc) a substitution of the methionine at the position corresponding toposition M202 of the mature polypeptide of SEQ ID NO: 1 with any otheramino acid, wherein the resulting variant has at least 80%, such as atleast 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 1 or 2; and recovering the variant.

The variants may be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis may be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis may also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parentalpha-amylase and subsequent ligation of an oligonucleotide containingthe mutation in the polynucleotide. Usually the restriction enzyme thatdigests the plasmid and the oligonucleotide is the same, permittingsticky ends of the plasmid and the insert to ligate to one another. See,e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955;and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis may also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure may be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis may be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions may be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods may be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides may be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga variant of the present invention. Accordingly, the present inventionrelates to isolated polynucleotides encoding a variant comprising a) adeletion at two or more positions corresponding to positions R181, G182,D183 and G184 of the mature polypeptide of SEQ ID NO: 1, b) asubstitution at one or more positions corresponding to positions Y198,Y200, L201, Y203, and A204 of the mature polypeptide of SEQ ID NO: 1,and c) substitution of the methionine at the position corresponding toposition M202 of the mature polypeptide of SEQ ID NO: 1, wherein thevariant has at least 80%, such as at least 90%, but less than 100%sequence identity with the mature polypeptide of SEQ ID NO: 1 or 2, andwherein the variant has alpha-amylase activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences. Accordingly, the present invention relatesto nucleic acid constructs comprising a polynucleotide encoding avariant comprising a) a deletion at two or more positions correspondingto positions R181, G182, D183 and G184 of the mature polypeptide of SEQID NO: 1, b) a substitution at one or more positions corresponding topositions Y198, Y200, L201, Y203, and A204 of the mature polypeptide ofSEQ ID NO: 1, and c) substitution of the methionine at the positioncorresponding to position M202 of the mature polypeptide of SEQ ID NO:1, wherein the variant has at least 80%, such as at least 90%, but lessthan 100% sequence identity with the mature polypeptide of SEQ ID NO: 1or 2, and wherein the variant has alpha-amylase activity, wherein thepolynucleotide is operably linked to one or more control sequences thatdirect the expression of the coding sequence in a suitable host cellunder conditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well-known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-alpha-amylase gene (amyQ), Bacillus licheniformisalpha-alpha-amylase gene (amyL), Bacillus licheniformis penicillinasegene (penP), Bacillus stearothermophilus maltogenic alpha-amylase gene(amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilisxylA and xylB genes, Bacillus thuringiensis crylIIA gene (Agaisse andLereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon,E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomycescoelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731),as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci.USA 80: 21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-alpha-amylase, Aspergillusniger acid stable alpha-alpha-amylase, Aspergillus niger or Aspergillusawamori glucoalpha-amylase (glaA), Aspergillus oryzae TAKAalpha-amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusariumvenenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900),Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase,Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanaseIV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a modified promoter from an Aspergillusneutral alpha-alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus triosephosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-alpha-amylase gene inwhich the untranslated leader has been replaced by an untranslatedleader from an Aspergillus nidulans or Aspergillus oryzae triosephosphate isomerase gene); and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-alpha-amylase (amyL), and Escherichia coli ribosomalRNA (rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoalpha-amylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA alpha-amylase, and Fusariumoxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis crylIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA alpha-amylase and Aspergillusnidulans triose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence; a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoalpha-amylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA alpha-amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently comprise a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may comprise a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCI B 11837 maltogenic alpha-amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral alpha-amylase, Aspergillus nigerglucoalpha-amylase, Aspergillus oryzae TAKA alpha-amylase, Humicolainsolens cellulase, Humicola insolens endoglucanase V, Humicolalanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and maybe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoalpha-amylase promoter,Aspergillus oryzae TAKA alpha-alpha-amylase promoter, and Aspergillusoryzae glucoalpha-amylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promotor, and transcriptional and translational stop signals.Accordingly, the present invention relates to a recombinant expressionvector comprising a polynucleotide encoding a variant comprising a) adeletion at two or more positions corresponding to positions R181, G182,D183 and G184 of the mature polypeptide of SEQ ID NO: 1, b) asubstitution at one or more positions corresponding to positions Y198,Y200, L201, Y203, and A204 of the mature polypeptide of SEQ ID NO: 1,and c) substitution of the methionine at the position corresponding toposition M202 of the mature polypeptide of SEQ ID NO: 1, wherein thevariant has at least 80%, such as at least 90%, but less than 100%sequence identity with the mature polypeptide of SEQ ID NO: 1 or 2, andwherein the variant has alpha-amylase activity, and wherein the vectorfurther comprises a promotor, and transcriptional and translational stopsignals.

The various nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may comprise any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercomprise the total DNA to be introduced into the genome of the hostcell, or a transposon, may be used.

The vector preferably comprises one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably comprises an element(s) that permits integrationof the vector into the host cell's genome or autonomous replication ofthe vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may compriseadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should comprise asufficient number of nucleic acids, such as 100 to 10,000 base pairs,400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have ahigh degree of sequence identity to the corresponding target sequence toenhance the probability of homologous recombination. The integrationalelements may be any sequence that is homologous with the target sequencein the genome of the host cell. Furthermore, the integrational elementsmay be non-encoding or encoding polynucleotides. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genemay be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide may be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells comprising amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, may be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention arewell-known to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

The alpha-amylase variants of the present invention may be expressed asdescribed in WO2010/115021.

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. Accordingly, the present inventionrelates to recombinant host cells, comprising a polynucleotide encodinga variant comprising a) a deletion at two or more positionscorresponding to positions R181, G182, D183 and G184 of the maturepolypeptide of SEQ ID NO: 1, b) a substitution at one or more positionscorresponding to positions Y198, Y200, L201, Y203, and A204 of themature polypeptide of SEQ ID NO: 1, and c) substitution of themethionine at the position corresponding to position M202 of the maturepolypeptide of SEQ ID NO: 1, wherein the variant has at least 80%, suchas at least 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 1 or 2, and wherein the variant hasalpha-amylase activity, wherein the polynucleotide is operably linked toone or more control sequences that direct the production of the variant.

A construct or vector comprising a polynucleotide is introduced into ahost cell so that the construct or vector is maintained as a chromosomalintegrant or as a self-replicating extra-chromosomal vector as describedearlier. The term “host cell” encompasses any progeny of a parent cellthat is not identical to the parent cell due to mutations that occurduring replication. The choice of a host cell will to a large extentdepend upon the gene encoding the variant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chtysosporium queenslandicum, Chtysosporium tropicum, Chtysosporiumzonaturn, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenaturn, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a variant,comprising: (a) cultivating a host cell of the present invention underconditions suitable for expression of the variant; and (b) recoveringthe variant. Accordingly, the present invention relates to methods ofproducing a variant comprising a) a deletion at two or more positionscorresponding to positions R181, G182, D183 and G184 of the maturepolypeptide of SEQ ID NO: 1, b) a substitution at one or more positionscorresponding to positions Y198, Y200, L201, Y203, and A204 of themature polypeptide of SEQ ID NO: 1, and c) substitution of themethionine at the position corresponding to position M202 of the maturepolypeptide of SEQ ID NO: 1, wherein the variant has at least 80%, suchas at least 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 1 or 2, and wherein the variant hasalpha-amylase activity, wherein the method comprises (a) cultivating ahost cell expressing the variant under conditions suitable forexpression of the variant; and (b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant may be recovered directly from the medium. If the variant is notsecreted, it may be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Compositions

The present invention also relates to compositions comprising a variantof the present invention. Accordingly, the present invention relates tocompositions comprising a variant comprising a) a deletion at two ormore positions corresponding to positions R181, G182, D183 and G184 ofthe mature polypeptide of SEQ ID NO: 1, b) a substitution at one or morepositions corresponding to positions Y198, Y200, L201, Y203, and A204 ofthe mature polypeptide of SEQ ID NO: 1, and c) substitution of themethionine at the position corresponding to position M202 of the maturepolypeptide of SEQ ID NO: 1, wherein the variant has at least 80%, suchas at least 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 1 or 2, and wherein the variant hasalpha-amylase activity.

Preferably, the compositions are enriched in such a variant. The term“enriched” means that the alpha-amylase activity of the composition hasbeen increased, e.g., with an enrichment factor of 1.1.

The composition may comprise a variant as the major enzymatic component,e.g., a mono-component composition. Alternatively, the composition maycomprise multiple enzymatic activities, such as an aminopeptidase,amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase,lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase,peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, or xylanase. The additional enzyme(s)may be produced, for example, by a microorganism belonging to the genusAspergillus, e.g., Aspergillus aculeatus, Aspergillus awamori,Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae;Fusarium, e.g., Fusarium bactridioides, Fusarium cerealis, Fusariumcrookwellense, Fusarium culmorum, Fusarium graminearum, Fusariumgraminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,Fusarium reticulaturn, Fusarium roseum, Fusarium sambucinum, Fusariumsarcochroum, Fusarium sulphureum, Fusarium toruloseum, Fusariumtrichothecioides, or Fusarium venenatum; Humicola, e.g., Humicolainsolens or Humicola lanuginosa; or Trichoderma, e.g., Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiaturn,Trichoderma reesei, or Trichoderma viride.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Forinstance, the composition may be in the form of a granulate or amicrogranulate. The variant may be stabilized in accordance with methodsknown in the art.

Detergent Compositions

In one embodiment, the invention is directed to detergent compositionscomprising an alpha-amylase variant of the present invention incombination with one or more additional cleaning composition components.Accordingly, the present invention relates to detergent compositionscomprising a variant comprising a) a deletion at two or more positionscorresponding to positions R181, G182, D183 and G184 of the maturepolypeptide of SEQ ID NO: 1, b) a substitution at one or more positionscorresponding to positions Y198, Y200, L201, Y203, and A204 of themature polypeptide of SEQ ID NO: 1, and c) substitution of themethionine at the position corresponding to position M202 of the maturepolypeptide of SEQ ID NO: 1, wherein the variant has at least 80%, suchas at least 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 1 or 2, and wherein the variant hasalpha-amylase activity, and wherein the detergent compositions furthercomprises one or more additional cleaning composition components.

The choice of additional components is within the skill of the artisanand includes conventional ingredients, including the exemplarynon-limiting components set forth below. The choice of components mayinclude, for fabric care, the consideration of the type of fabric to becleaned, the type and/or degree of soiling, the temperature at whichcleaning is to take place, and the formulation of the detergent product.Although components mentioned below are categorized by general headeraccording to a particular functionality, this is not to be construed asa limitation, as a component may comprise additional functionalities aswill be appreciated by the skilled artisan.

Concentration of the Enzyme of the Present Invention

In one embodiment of the present invention, the polypeptide of thepresent invention may be used in the dishwashing composition in anamount corresponding to 0.001-200 mg of protein, such as 0.005-100 mg ofprotein, preferably 0.01-50 mg of protein, more preferably 0.05-20 mg ofprotein, even more preferably 0.1-10 mg of protein per liter of washliquor.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g. a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g. an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example,WO92/19709 and WO92/19708.

A polypeptide of the present invention may also be incorporated in thedetergent formulations disclosed in WO97/07202, which is herebyincorporated by reference.

Surfactants

The dish washing composition may comprise at least one non-ionicsurfactant. Suitable nonionic surfactants include, but are not limitedto low-foaming nonionic (LFNI) surfactants. An LFNI surfactant is mosttypically used in an automatic dishwashing composition because of theimproved water-sheeting action (especially from glassware) which theyconfer to the automatic dishwashing composition. They also may encompassnon-silicone, phosphate or nonphosphate polymeric materials which areknown to defoam food soils encountered in automatic dishwashing. TheLFNI surfactant may have a relatively low cloud point and a highhydrophilic-lipophilic balance (HLB). Cloud points of 1% solutions inwater are typically below about 32° C. alternatively lower, e.g., 0° C.,for optimum control of sudsing throughout a full range of watertemperatures. If desired, a biodegradable LFNI surfactant having theabove properties may be used.

A LFNI surfactant may include, but is not limited to: alkoxylatedsurfactants, especially ethoxylates derived from primary alcohols, andblends thereof with more sophisticated surfactants, such as thepolyoxypropylene/polyoxyethylene/polyoxypropylene reverse blockpolymers. Suitable block polyoxyethylene-polyoxypropylene polymericcompounds that meet the requirements may include those based on ethyleneglycol, propylene glycol, glycerol, trimethylolpropane andethylenediamine, and mixtures thereof. Polymeric compounds made from asequential ethoxylation and propoxylation of initiator compounds with asingle reactive hydrogen atom, such as C 12—is aliphatic alcohols, donot generally provide satisfactory suds control in Automatic dishwashingcompositions. However, certain of the block polymer surfactant compoundsdesignated as PLURONIC(R) and TETRONIC(R) by the BASF-Wyandotte Corp.,Wyandotte, Mich., are suitable in Automatic dishwashing compositions.The LFNI surfactant may optionally include a propylene oxide in anamount up to about 15% by weight. Other LFNI surfactants may be preparedby the processes described in U.S. Pat. No. 4,223,163. The LFNIsurfactant may also be derived from a straight chain fatty alcoholcontaining from about 16 to about 20 carbon atoms (C16-C20 alcohol),alternatively a Ci8 alcohol, condensed with an average of from about 6to about 15 moles, or from about 7 to about 12 moles, and alternatively,from about 7 to about 9 moles of ethylene oxide per mole of alcohol. Theethoxylated nonionic surfactant so derived may have a narrow ethoxylatedistribution relative to the average.

In certain embodiments, an LFNI surfactant having a cloud point below30° C. may be present in an amount from about 0.01% to about 60%, orfrom about 0.5% to about 10% by weight, and alternatively, from about 1%to about 5% by weight of the composition.

In preferred embodiments, the surfactant is a non-ionic surfactant or anon-ionic surfactant system having a phase inversion temperature, asmeasured at a concentration of 1% in distilled water, between 40 and 70°C., preferably between 45 and 65° C. By a “non-ionic surfactant system”is meant herein a mixture of two or more non-ionic surfactants.Preferred for use herein are non-ionic surfactant systems. They seem tohave improved cleaning and finishing properties and stability in productthan single non-ionic surfactants. Suitable nonionic surfactantsinclude: i) ethoxylated non-ionic surfactants prepared by the reactionof a monohydroxy alkanol or alkyphenol with 6 to 20 carbon atoms withpreferably at least 12 moles particularly preferred at least 16 moles,and still more preferred at least 20 moles of ethylene oxide per mole ofalcohol or alkylphenol; ii) alcohol alkoxylated surfactants having from6 to 20 carbon atoms and at least one ethoxy and propoxy group.Preferred for use herein are mixtures of surfactants i) and ii).

Another suitable non-ionic surfactants are epoxy-cappedpoly(oxyalkylated) alcohols represented by the formula:R₁O[CH₂CH(CH₃)O]_(x)[CH₂CH₂O]_(y)[CH₂CH(OH)R₂]  (I)

wherein R₁ is a linear or branched, aliphatic hydrocarbon radical havingfrom 4 to 18 carbon atoms; R₂ is a linear or branched aliphatichydrocarbon radical having from 2 to 26 carbon atoms; x is an integerhaving an average value of from 0.5 to 1.5, more preferably about 1; andy is an integer having a value of at least 15, more preferably at least20. Preferably, the surfactant of formula I has at least about 10 carbonatoms in the terminal epoxide unit [CH₂CH(OH)R₂]. Suitable surfactantsof formula I are Olin Corporation's POLY-TERGENT(R) SLF-18B nonionicsurfactants, as described, for example, in WO 94/22800, published Oct.13, 1994 by Olin Corporation.

Preferably, non-ionic surfactants and/or system herein have a Draveswetting time of less than 360 seconds, preferably less than 200 seconds,more preferably less than 100 seconds and especially less than 60seconds as measured by the Draves wetting method (standard method ISO8022 using the following conditions; 3-g hook, 5-g cotton skein, 0.1% byweight aqueous solution at a temperature of 25° C.). Amine oxidessurfactants are also useful in the present invention asanti-redeposition surfactants include linear and branched compoundshaving the formula:

wherein R³ is selected from an alkyl, hydroxyalkyl, acylamidopropoyl andalkyl phenyl group, or mixtures thereof, containing from 8 to 26 carbonatoms, preferably 8 to 18 carbon atoms; R⁴ is an alkylene orhydroxyalkylene group containing from 2 to 3 carbon atoms, preferably 2carbon atoms, or mixtures thereof; x is from 0 to 5, preferably from 0to 3; and each R⁵ is an alkyl or hydroxyalkyl group containing from 1 to3, preferably from 1 to 2 carbon atoms, or a polyethylene oxide groupcontaining from 1 to 3, preferable 1, ethylene oxide groups. The R⁵groups may be attached to each other, e.g., through an oxygen ornitrogen atom, to form a ring structure.

These amine oxide surfactants in particular include C₁₀-C₁₈ alkyldimethyl amine oxides and C₈-C₁₈ alkoxy ethyl dihydroxyethyl amineoxides. Examples of such materials include dimethyloctylamine oxide,diethyldecylamine oxide, bis-(2-hydroxyethyl)dodecylamine oxide,dimethyldodecylamine oxide, dipropyltetradecylamine oxide,methylethylhexadecylamine oxide, dodecylamidopropyl dimethylamine oxide,cetyl dimethylamine oxide, stearyl dimethylamine oxide, tallowdimethylamine oxide and dimethyl-2-hydroxyoctadecylamine oxide.Preferred are C₁₀-C₁₈ alkyl dimethylamine oxide, and C₁₀-C₁₈ acylamidoalkyl dimethylamine oxide. Surfactants and especially non-ionicsurfactants may be present in amounts from 0 to 10% by weight,preferably from 0.1% to 10%, and most preferably from 0.25% to 6%.

Sulfonated Polymer

The polymer, if used, is used in any suitable amount from about 0.1% toabout 20%, preferably from 1% to about 15%, more preferably from 2% to10% by weight of the composition. Sulfonated/carboxylated polymers areparticularly suitable for the compositions contained in a pouch.

Suitable sulfonated/carboxylated polymers described herein may have aweight average molecular weight of less than or equal to about 100,000Da, or less than or equal to about 75,000 Da, or less than or equal toabout 50,000 Da, or from about 3,000 Da to about 50,000, preferably fromabout 5,000 Da to about 45,000 Da.

As noted herein, the sulfonated/carboxylated polymers may comprise (a)at least one structural unit derived from at least one carboxylic acidmonomer having the general formula (I):

wherein R¹ to R⁴ are independently hydrogen, methyl, carboxylic acidgroup or CH₂COOH and wherein the carboxylic acid groups may beneutralized; (b) optionally, one or more structural units derived fromat least one nonionic monomer having the general formula (II):

wherein R⁵ is hydrogen, C₁ to C₆ alkyl, or C₁ to C₆ hydroxyalkyl, and Xis either aromatic (with R⁵ being hydrogen or methyl when X is aromatic)or X is of the general formula (III):

wherein R⁶ is (independently of R⁵) hydrogen, C₁ to C₆ alkyl, or C₁ toC₆ hydroxyalkyl, and Y is O or N; and at least one structural unitderived from at least one sulfonic acid monomer having the generalformula (IV):

wherein R⁷ is a group comprising at least one sp² bond, A is O, N, P, Sor an amido or ester linkage, B is a mono- or polycyclic aromatic groupor an aliphatic group, each t is independently 0 or 1, and M⁺ is acation. In one aspect, R⁷ is a C₂ to C₆ alkene. In another aspect, R⁷ isethene, butene or propene.

Preferred carboxylic acid monomers include one or more of the following:acrylic acid, maleic acid, itaconic acid, methacrylic acid, orethoxylate esters of acrylic acids, acrylic and methacrylic acids beingmore preferred. Preferred sulfonated monomers include one or more of thefollowing: sodium (meth) allyl sulfonate, vinyl sulfonate, sodium phenyl(meth) allyl ether sulfonate, or 2-acrylamido-methyl propane sulfonicacid. Preferred non-ionic monomers include one or more of the following:methyl (meth) acrylate, ethyl (meth) acrylate, t-butyl (meth) acrylate,methyl (meth) acrylamide, ethyl (meth) acrylamide, t-butyl (meth)acrylamide, styrene, or [alpha]-methyl styrene.

Preferably, the polymer comprises the following levels of monomers: fromabout 40 to about 90%, preferably from about 60 to about 90% by weightof the polymer of one or more carboxylic acid monomer; from about 5 toabout 50%, preferably from about 10 to about 40% by weight of thepolymer of one or more sulfonic acid monomer; and optionally from about1% to about 30%, preferably from about 2 to about 20% by weight of thepolymer of one or more non-ionic monomer. An especially preferredpolymer comprises about 70% to about 80% by weight of the polymer of atleast one carboxylic acid monomer and from about 20% to about 30% byweight of the polymer of at least one sulfonic acid monomer.

The carboxylic acid is preferably (meth)acrylic acid. The sulfonic acidmonomer is preferably one of the following: 2-acrylamidomethyl-1-propanesulfonic acid,2-methacrylamido-2-methyl-1-propanesulfonic acid,3-methacrylamido-2-hydroxypropanesulfonic acid, allysulfonic acid,methallysulfonic acid, allyloxybenzenesulfonic acid,methallyloxybenzensulfonic acid,2-hydroxy-3-(2-propenyloxy)propanesulfonic acid,2-methyl-2-propene-I-sulfonic acid, styrene sulfonic acid, vinylsulfonicacid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate,sulfomethylacrylamid, sulfomethylmethacrylamide, and water soluble saltsthereof. The unsaturated sulfonic acid monomer is most preferably2-acrylamido-2-propanesulfonic acid (AMPS).

Preferred commercial available polymers include: Alcosperse 240,Aquatreat AR 540 and Aquatreat MPS supplied by Alco Chemical; Acumer3100, Acumer 2000, Acusol 587G and Acusol 588G supplied by Rohm & Haas;Goodrich K-798, K-775 and K-797 supplied by BF Goodrich; and ACP 1042supplied by ISP technologies Inc. Particularly preferred polymers areAcusol 587G and Acusol 588G supplied by Rohm & Haas.

In the polymers, all or some of the carboxylic or sulfonic acid groupsmay be present in neutralized form, i.e. the acidic hydrogen atom of thecarboxylic and/or sulfonic acid group in some or all acid groups may bereplaced with metal ions, preferably alkali metal ions and in particularwith sodium ions.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds inaqueous solutions (or oppositely, polar substances in a non-polarenvironment). Typically, hydrotropes have both hydrophilic and ahydrophobic character (so-called amphiphilic properties as known fromsurfactants); however the molecular structure of hydrotropes generallydo not favor spontaneous self-aggregation, see e.g. review by Hodgdonand Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not display a critical concentration above whichself-aggregation occurs as found for surfactants and lipids formingmiceller, lamellar or other well defined meso-phases. Instead, manyhydrotropes show a continuous-type aggregation process where the sizesof aggregates grow as concentration increases. However, many hydrotropesalter the phase behavior, stability, and colloidal properties of systemscomprising substances of polar and non-polar character, includingmixtures of water, oil, surfactants, and polymers. Hydrotropes areclassically used across industries from pharma, personal care, food, totechnical applications. Use of hydrotropes in detergent compositionsallow for example more concentrated formulations of surfactants (as inthe process of compacting liquid detergents by removing water) withoutinducing undesired phenomena such as phase separation or high viscosity.

The detergent may comprise 0-10% by weight, for example 0-5% by weight,such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope.Any hydrotrope known in the art for use in detergents may be utilized.Non-limiting examples of hydrotropes include sodium benzenesulfonate,sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodiumcumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcoholsand polyglycolethers, sodium hydroxynaphthoate, sodiumhydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, andcombinations thereof.

Builders and Co-Builders

The detergent composition may comprise about 0-65% by weight, such asabout 5% to about 50% of a detergent builder or co-builder, or a mixturethereof. In a dish wash detergent, the level of builder is typically40-65%, particularly 50-65%. The builder and/or co-builder mayparticularly be a chelating agent that forms water-soluble complexeswith Ca and Mg. Any builder and/or co-builder known in the art for usein ADW detergents may be utilized. Non-limiting examples of buildersinclude zeolites, diphosphates (pyrophosphates), triphosphates such assodium triphosphate (STP or STPP), carbonates such as sodium carbonate,soluble silicates such as sodium metasilicate, layered silicates (e.g.,SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA),diethanolamine (DEA, also known as 2,2′-iminodiethan-1-ol),triethanolamine (TEA, also known as 2,2′,2″-nitrilotriethan-1-ol), and(carboxymethyl)inulin (CMI), and combinations thereof.

The detergent composition may also comprise 0-50% by weight, such asabout 5% to about 30%, of a detergent co-builder. The detergentcomposition may include a co-builder alone, or in combination with abuilder, for example a zeolite builder. Non-limiting examples ofco-builders include homopolymers of polyacrylates or copolymers thereof,such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid)(PAA/PMA). Further non-limiting examples include citrate, chelators suchas aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl-or alkenylsuccinic acid. Additional specific examples include2,2′,2″-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid(IDS), ethylenediamine-N,N′-disuccinic acid (EDDS),methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid(GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP),ethylenediaminetetra(methylenephosphonic acid) (EDTMPA),diethylenetriaminepentakis(methylenephosphonic acid) (DTMPA or DTPMPA),N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoaceticacid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), asparticacid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA),N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid(SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL),N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid(MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid(SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diaceticacid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) andsulfomethyl-N,N-diacetic acid (SMDA),N-(2-hydroxyethyl)ethylenediamine-N,N′,N″-triacetic acid (HEDTA),diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonicacid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), andcombinations and salts thereof. Further exemplary builders and/orco-builders are described in, e.g., WO 09/102854, U.S. Pat. No.5,977,053

Bleaching Systems

Inorganic and organic bleaches are suitable cleaning actives for useherein. Inorganic bleaches include perhydrate salts such as perborate,percarbonate, perphosphate, persulfate and persilicate salts. Theinorganic perhydrate salts are normally the alkali metal salts. Theinorganic perhydrate salt may be included as the crystalline solidwithout additional protection. Alternatively, the salt may be coated.

Alkali metal percarbonates, particularly sodium percarbonate arepreferred perhydrates for use herein. The percarbonate is mostpreferably incorporated into the products in a coated form whichprovides in-product stability. A suitable coating material providing inproduct stability comprises mixed salt of a water-soluble alkali metalsulphate and carbonate. Such coatings together with coating processeshave previously been described in GB 1,466,799. The weight ratio of themixed salt coating material to percarbonate lies in the range from 1:200to 1:4, more preferably from 1:99 to 1:9, and most preferably from 1:49to 1:19. Preferably, the mixed salt is of sodium sulphate and sodiumcarbonate which has the general formula Na2S04.n.Na2CO3 wherein n isfrom 0.1 to 3, preferably n is from 0.3 to 1.0 and most preferably n isfrom 0.2 to 0.5.

Another suitable coating material providing in product stability,comprises sodium silicate of SiO2: Na20 ratio from 1.8:1 to 3.0:1,preferably 1.8:1 to 2.4:1, and/or sodium metasilicate, preferablyapplied at a level of from 2% to 10%, (normally from 3% to 5%) of SiO2by weight of the inorganic perhydrate salt. Magnesium silicate may alsobe included in the coating. Coatings that comprise silicate and boratesalts or boric acids or other inorganics are also suitable.

Other coatings which contain waxes, oils, fatty soaps can also be usedadvantageously within the present invention.

Potassium peroxymonopersulfate is another inorganic perhydrate salt ofutility herein. Typical organic bleaches are organic peroxyacidsincluding diacyl and tetraacylperoxides, especially diperoxydodecanediocacid, diperoxytetradecanedioc acid, and diperoxyhexadecanedioc acid.Dibenzoyl peroxide is a preferred organic peroxyacid herein. Mono- anddiperazelaic acid, mono- and diperbrassylic acid, andNphthaloylaminoperoxicaproic acid are also suitable herein. The diacylperoxide, especially dibenzoyl peroxide, should preferably be present inthe form of particles having a weight average diameter of from about 0.1to about 100 microns, preferably from about 0.5 to about 30 microns,more preferably from about 1 to about 10 microns. Preferably, at leastabout 25%, more preferably at least about 50%, even more preferably atleast about 75%, most preferably at least about 90%, of the particlesare smaller than 10 microns, preferably smaller than 6 microns. Diacylperoxides within the above particle size range have also been found toprovide better stain removal especially from plastic dishware, whileminimizing undesirable deposition and filming during use in automaticdishwashing machines, than larger diacyl peroxide particles. Thepreferred diacyl peroxide particle size thus allows the formulator toobtain good stain removal with a low level of diacyl peroxide, whichreduces deposition and filming. Conversely, as diacyl peroxide particlesize increases, more diacyl peroxide is needed for good stain removal,which increases deposition on surfaces encountered during thedishwashing process.

Further typical organic bleaches include the peroxy acids, particularexamples being the alkylperoxy acids and the arylperoxy acids. Preferredrepresentatives are (a) peroxybenzoic acid and its ring-substitutedderivatives, such as alkylperoxybenzoic acids, but alsoperoxy-[alpha]-naphthoic acid and magnesium monoperphthalate, (b) thealiphatic or substituted aliphatic peroxy acids, such as peroxylauricacid, peroxystearic acid, [epsilon]-phthalimidoperoxycaproicacid[phthaloiminoperoxyhexanoic acid (PAP)],o-carboxybenzamidoperoxycaproic acid, N-nonenylamidoperadipic acid andN-nonenylamidopersuccinates, and (c) aliphatic and araliphaticperoxydicarboxylic acids, such as 1,12-diperoxycarboxylic acid,1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid,the diperoxyphthalic acids, 2-decyldiperoxybutane-1,4-dioic acid,N,N-terephthaloyldi(6-aminopercaproic acid).

Bleach Activators

Bleach activators are typically organic peracid precursors that enhancethe bleaching action in the course of cleaning at temperatures of 60° C.and below. Bleach activators suitable for use herein include compoundswhich, under perhydrolysis conditions, give aliphatic peroxoycarboxylicacids having preferably from 1 to 10 carbon atoms, in particular from 2to 4 carbon atoms, and/or optionally substituted perbenzoic acid.Suitable substances bear O-acyl and/or N-acyl groups of the number ofcarbon atoms specified and/or optionally substituted benzoyl groups.Preference is given to polyacylated alkylenediamines, in particulartetraacetylethylenediamine (TAED), acylated triazine derivatives, inparticular I,5-diacetyl-2,4-dioxohexahydro-I,3,5-triazine (DADHT),acylated glycolurils, in particular tetraacetylglycoluril (TAGU),N-acylimides, in particular N-nonanoylsuccinimide (NOSI), acylatedphenolsulfonates, in particular n-nonanoyl- orisononanoyloxybenzenesulfonate (n- or iso-NOBS), carboxylic anhydrides,in particular phthalic anhydride, acylated polyhydric alcohols, inparticular triacetin, ethylene glycol diacetate and2,5-diacetoxy-2,5-dihydrofuran and also triethylacetyl citrate (TEAC).Bleach activators if included in the compositions of the invention arein a level of from about 0.1 to about 10%, preferably from about 0.5 toabout 2% by weight of the composition.

Bleach Catalyst

Bleach catalysts preferred for use herein include the manganesetriazacyclononane and related complexes (U.S. Pat. Nos. 4,246,612,5,227,084); Co, Cu, Mn and Fe bispyridylamine and related complexes(U.S. Pat. No. 5,114,611); and pentamine acetate cobalt(III) and relatedcomplexes (U.S. Pat. No. 4,810,410). A complete description of bleachcatalysts suitable for use herein can be found in WO 99/06521, pages 34,line 26 to page 40, line 16. Bleach catalyst if included in thecompositions of the invention are in a level of from about 0.1 to about10%, preferably from about 0.5 to about 2% by weight of the composition.

Oxidoreductases, for example oxidases, oxygenases, catalases,peroxidases such as halo-, chloro-, bromo-, lignin, glucose, ormanganese peroxidases, dioxygenases, or laccases (phenoloxidases,polyphenoloxidases), may also be used according to the present inventionto intensify the bleaching effect. Advantageously, preferably organic,particularly preferably aromatic compounds that interact with theenzymes are additionally added in order to enhance the activity of therelevant oxidoreductases (enhancers) or, if there is a large differencein redox potentials between the oxidizing enzymes and the stains, toensure electron flow (mediators).

Silicates

Preferred silicates are sodium silicates such as sodium disilicate,sodium metasilicate and crystalline phyllosilicates. Silicates ifpresent are at a level of from about 1 to about 20%, preferably fromabout 5 to about 15% by weight of composition.

Metal Care Agents

Metal care agents may prevent or reduce the tarnishing, corrosion oroxidation of metals, including aluminium, stainless steel andnon-ferrous metals, such as silver and copper. Suitable examples includeone or more of the following:

(a) benzatriazoles, including benzotriazole or bis-benzotriazole andsubstituted derivatives thereof. Benzotriazole derivatives are thosecompounds in which the available substitution sites on the aromatic ringare partially or completely substituted. Suitable substituents includelinear or branch-chain Ci-C20-alkyl groups and hydroxyl, thio, phenyl orhalogen such as fluorine, chlorine, bromine and iodine.

(b) metal salts and complexes chosen from the group consisting of zinc,manganese, titanium, zirconium, hafnium, vanadium, cobalt, gallium andcerium salts and/or complexes, the metals being in one of the oxidationstates II, III, IV, V or VI. In one aspect, suitable metal salts and/ormetal complexes may be chosen from the group consisting of Mn(II)sulphate, Mn(II) citrate, Mn(II) stearate, Mn(II) acetylacetonate,K{circumflex over ( )}ATiF6, K{circumflex over ( )}ZrF6, CoSO4, Co(NOs)2and Ce(NOs)3, zinc salts, for example zinc sulphate, hydrozincite orzinc acetate; (c) silicates, including sodium or potassium silicate,sodium disilicate, sodium metasilicate, crystalline phyllosilicate andmixtures thereof.

Further suitable organic and inorganic redox-active substances that actas silver/copper corrosion inhibitors are disclosed in WO 94/26860 andWO 94/26859. Preferably the composition of the invention comprises from0.1 to 5% by weight of the composition of a metal care agent, preferablythe metal care agent is a zinc salt.

Enzymes

The detergent additive as well as the detergent composition may compriseone or more additional enzymes such as a protease, lipase, cutinase, anamylase, carbohydrase, cellulase, pectinase, mannanase, arabinase,galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

In general, the properties of the selected enzyme(s) should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzyme(s) should be present in effective amounts.

Cellulases

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178,5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and WO099/001544.

Other cellulases are endo-beta-1,4-glucanase enzyme having a sequence ofat least 97% identity to the amino acid sequence of position 1 toposition 773 of SEQ ID NO:2 of WO 2002/099091 or a family 44xyloglucanase, which a xyloglucanase enzyme having a sequence of atleast 60% identity to positions 40-559 of SEQ ID NO: 2 of WO2001/062903.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S) Carezyme Premium™ (Novozymes A/S), Celluclean™(Novozymes A/S), Celluclean Classic™ (Novozymes A/S), Cellusoft™(Novozymes A/S), Whitezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™(Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

Proteases

Suitable proteases include those of bacterial, fungal, plant, viral oranimal origin e.g. vegetable or microbial origin. Microbial origin ispreferred. Chemically modified or protein engineered variants areincluded. It may be an alkaline protease, such as a serine protease or ametalloprotease. A serine protease may for example be of the S1 family,such as trypsin, or the S8 family such as subtilisin. A metalloproteaseprotease may for example be a thermolysin from e.g. family M4 or othermetalloprotease such as those from M5, M7 or M8 families.

The term “subtilases” refers to a sub-group of serine protease accordingto Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al.Protein Science 6 (1997) 501-523. Serine proteases are a subgroup ofproteases characterized by having a serine in the active site, whichforms a covalent adduct with the substrate. The subtilases may bedivided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitasefamily, the Proteinase K family, the Lantibiotic peptidase family, theKexin family and the Pyrolysin family.

Examples of subtilases are those derived from Bacillus such as Bacilluslentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacilluspumilus and Bacillus gibsonii described in; U.S. Pat. No. 7,262,042 andWO09/021867, and subtilisin lentus, subtilisin Novo, subtilisinCarlsberg, Bacillus licheniformis, subtilisin BPN′, subtilisin 309,subtilisin 147 and subtilisin 168 described in WO89/06279 and proteasePD138 described in (WO93/18140). Other useful proteases may be thosedescribed in WO92/175177, WO01/016285, WO02/026024 and WO02/016547.Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO89/06270,WO94/25583 and WO05/040372, and the chymotrypsin proteases derived fromCellumonas described in WO05/052161 and WO05/052146.

A further preferred protease is the alkaline protease from Bacilluslentus DSM 5483, as described for example in WO95/23221, and variantsthereof which are described in WO92/21760, WO95/23221, EP1921147 andEP1921148.

Examples of metalloproteases are the neutral metalloprotease asdescribed in WO07/044993 (Genencor Int.) such as those derived fromBacillus amyloliquefaciens.

Examples of useful proteases are the variants described in: WO92/19729,WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452,WO003/006602, WO04/03186, WO04/041979, WO07/006305, WO11/036263,WO11/036264, especially the variants with substitutions in one or moreof the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130,160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235,236, 245, 248, 252 and 274 using the BPN′ numbering. More preferred thesubtilase variants may comprise the mutations: S3T, V4I, S9R, A15T,K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,RS103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A,G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S,A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN′ numbering).

Suitable commercially available protease enzymes include those soldunder the trade names Alcalase®, Duralase®, Durazym®, Relase®, Relase®Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®,Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra,Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under thetradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®,Preferenz™, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®,Properase®, Effectenz™, FN2®, FN3®, FN4®, Excellase®, Opticlean® andOptimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.), BLAP (sequenceshown in FIG. 29 of U.S. Pat. No. 5,352,604) and variants hereof (HenkelAG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

Savinase® is marketed by NOVOZYMES A/S. It is subtilisin 309 from B.Lentus and differs from BAALKP only in one position (N87S).

Lipases and Cutinases

Suitable lipases and cutinases include those of bacterial or fungalorigin. Chemically modified or protein engineered variant enzymes areincluded. Examples include lipase from Thermomyces, e.g. from T.lanuginosus (previously named Humicola lanuginosa) as described inEP258068 and EP305216, cutinase from Humicola, e.g. H. insolens(WO96/13580), lipase from strains of Pseudomonas (some of these nowrenamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes(EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 &WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyceslipases (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560),cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipasefrom Thermobifida fusca (WO11/084412), Geobacillus stearothermophiluslipase (WO11/084417), lipase from Bacillus subtilis (WO11/084599), andlipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis(WO12/137147).

Other examples are lipase variants such as those described in EP407225,WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381,WO95/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063,WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial lipase products include include Lipolase™, Lipex™;Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally fromGenencor) and Lipomax (originally from Gist-Brocades).

Still other examples are lipases sometimes referred to asacyltransferases or perhydrolases, e.g. acyltransferases with homologyto Candida antarctica lipase A (WO10/111143), acyltransferase fromMycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family(WO09/67279), and variants of the M. smegmatis perhydrolase inparticular the S54V variant used in the commercial product Gentle PowerBleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

Additional Amylases

Suitable amylases which may be used together with the enzyme preparationof the invention may be an alpha-amylase, a pullulanase or aglucoamylase and may be of bacterial or fungal origin. Chemicallymodified or protein engineered variants are included. Amylases include,for example, alpha-amylases obtained from Bacillus, e.g., a specialstrain of Bacillus licheniformis, described in more detail in GB1,296,839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 orvariants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferredvariants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQID NO: 4 of WO 99/019467, such as variants with substitutions in one ormore of the following positions: 15, 23, 105, 106, 124, 128, 133, 154,156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243,264, 304, 305, 391, 408, and 444.

Different suitable amylases include amylases having SEQ ID NO: 6 in WO02/010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO: 6 are those having a deletion inpositions 181 and 182 and a substitution in position 193.

Other amylases which are suitable are hybrid alpha-amylase comprisingresidues 1-33 of the alpha-amylase derived from B. amyloliquefaciensshown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B.licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 orvariants having 90% sequence identity thereof. Preferred variants ofthis hybrid alpha-amylase are those having a substitution, a deletion oran insertion in one of more of the following positions: G48, T49, G107,H156, A181, N190, M197, I201, A209 and Q264. Most preferred variants ofthe hybrid alpha-amylase comprising residues 1-33 of the alpha-amylasederived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are those having thesubstitutions: M197T; H156Y+A181T+N190F+A209V+Q264S; orG48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Further amylases which are suitable are amylases having SEQ ID NO: 6 inWO 99/019467 or variants thereof having 90% sequence identity to SEQ IDNO: 6. Preferred variants of SEQ ID NO: 6 are those having asubstitution, a deletion or an insertion in one or more of the followingpositions: R181, G182, H183, G184, N195, I206, E212, E216 and K269.Particularly preferred amylases are those having deletion in positionsR181 and G182, or positions H183 and G184.

Additional amylases which may be used are those having SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variantsthereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, adeletion or an insertion in one or more of the following positions: 140,181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using SEQID 2 of WO 96/023873 for numbering. More preferred variants are thosehaving a deletion in two positions selected from 181, 182, 183 and 184,such as 181 and 182, 182 and 183, or positions 183 and 184. Mostpreferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7are those having a deletion in positions 183 and 184 and a substitutionin one or more of positions 140, 195, 206, 243, 260, 304 and 476.

Other amylases which may be used are amylases having SEQ ID NO: 2 of WO08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90%sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequenceidentity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ IDNO: 10 in WO 01/66712 are those having a substitution, a deletion or aninsertion in one of more of the following positions: 176, 177, 178, 179,190, 201, 207, 211 and 264.

Further suitable amylases are amylases having SEQ ID NO: 2 of WO09/061380 or variants having 90% sequence identity to SEQ ID NO: 2thereof. Preferred variants of SEQ ID NO: 2 are those having atruncation of the C-terminus and/or a substitution, a deletion or aninsertion in one of more of the following positions: Q87, Q98, S125,N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243,N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferredvariants of SEQ ID NO: 2 are those having the substitution in one ofmore of the following positions: Q87E,R, Q98R, S125A, N128C, T131I,T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R,R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180and/or S181 or of T182 and/or G183. Most preferred amylase variants ofSEQ ID NO: 2 are those having the substitutions:N128C+K178L+T182G+Y305R+G475K;N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;S125A+N128C+K178L+T182G+Y305R+G475K; orS125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variants areC-terminally truncated and optionally further comprises a substitutionat position 243 and/or a deletion at position 180 and/or position 181.

Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 inWO01/66712 or a variant having at least 90% sequence identity to SEQ IDNO: 12. Preferred amylase variants are those having a substitution, adeletion or an insertion in one of more of the following positions ofSEQ ID NO: 12 in WO01/66712: R28, R118, N174; R181, G182, D183, G184,G186, WO189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320,H324, E345, Y396, R400, WO439, R444, N445, K446, Q449, R458, N471, N484.Particular preferred amylases include variants having a deletion of D183and G184 and having the substitutions R118K, N195F, R320K and R458K, anda variant additionally having substitutions in one or more positionselected from the group: M9, G149, G182, G186, M202, T257, Y295, N299,M323, E345 and A339, most preferred a variant that additionally hassubstitutions in all these positions.

Other examples are amylase variants such as those described inWO2011/098531, WO2013/001078 and WO2013/001087.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™,Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (fromNovozymes A/S), and Rapidase™, Purastar™/Effectenz™, Powerase, PreferenzS100, Preferenz S110 and Preferenz S1000 (from Genencor InternationalInc./DuPont).

Peroxidases/Oxidases

A peroxidase is an enzyme comprised by the enzyme classification EC1.11.1.7, as set out by the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (IUBMB), or any fragmentderived therefrom, exhibiting peroxidase activity.

Suitable peroxidases include those of plant, bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful peroxidases include peroxidases from Coprinopsis, e.g., fromC. cinerea (EP 179,486), and variants thereof as those described in WO93/24618, WO 95/10602, and WO 98/15257.

A peroxidase also includes a haloperoxidase enzyme, such aschloroperoxidase, bromoperoxidase and compounds exhibitingchloroperoxidase or bromoperoxidase activity. Haloperoxidases areclassified according to their specificity for halide ions.Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochloritefrom chloride ions.

In an embodiment, the haloperoxidase is a chloroperoxidase. Preferably,the haloperoxidase is a vanadium haloperoxidase, i.e., avanadate-containing haloperoxidase, optionally wherein thevanadate-containing haloperoxidase is combined with a source of chlorideion.

Haloperoxidases have been isolated from many different fungi, inparticular from the fungus group dematiaceous hyphomycetes, such asCaldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C.verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.

Haloperoxidases have also been isolated from bacteria such asPseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S.aureofaciens.

In an preferred embodiment, the haloperoxidase is derivable fromCurvularia sp., in particular Curvularia verruculosa or Curvulariainaequalis, such as C. inaequalis CBS 102.42 as described in WO95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 asdescribed in WO 97/04102; or from Drechslera hartlebii as described inWO 01/79459, Dendryphiella salina as described in WO 01/79458,Phaeotrichoconis crotalarie as described in WO 01/79461, orGeniculosporium sp. as described in WO 01/79460.

An oxidase according to the invention include, in particular, anylaccase enzyme comprised by the enzyme classification EC 1.10.3.2, orany fragment derived therefrom exhibiting laccase activity, or acompound exhibiting a similar activity, such as a catechol oxidase (EC1.10.3.1), an o-aminophenol oxidase (EC 1.10.3.4), or a bilirubinoxidase (EC 1.3.3.5).

Preferred laccase enzymes are enzymes of microbial origin. The enzymesmay be derived from plants, bacteria or fungi (including filamentousfungi and yeasts).

Suitable examples from fungi include a laccase derivable from a strainof Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis,Collybia, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T.versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea,C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P.condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M.thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P.pinsitus, Phlebia, e.g., P. radiata (WO 92/01046), or Coriolus, e.g., C.hirsutus (JP 2238885).

Suitable examples from bacteria include a laccase derivable from astrain of Bacillus.

A laccase derived from Coprinopsis or Myceliophthora is preferred; inparticular a laccase derived from Coprinopsis cinerea, as disclosed inWO 97/08325; or from Myceliophthora thermophila, as disclosed in WO95/33836.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives comprising one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, maybe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g. as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1,000 to 20,000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

Adjunct Materials

Any detergent component known in the art for use in ADW detergents mayalso be utilized. Other optional detergent components includeanti-corrosion agents, anti-shrink agents, anti-soil redepositionagents, anti-wrinkling agents, bactericides, binders, corrosioninhibitors, disintegrants/disintegration agents, dyes, enzymestabilizers (including boric acid, borates, CMC, and/or polyols such aspropylene glycol), fabric conditioners including clays,fillers/processing aids, fluorescent whitening agents/opticalbrighteners, foam boosters, foam (suds) regulators, perfumes,soil-suspending agents, softeners, suds suppressors, tarnish inhibitors,and wicking agents, either alone or in combination. Any ingredient knownin the art for use ADW detergents may be utilized. The choice of suchingredients is well within the skill of the artisan.

Dispersants

The detergent compositions of the present invention may also comprisedispersants. In particular, powdered detergents may comprisedispersants. Suitable water-soluble organic materials include the homo-or co-polymeric acids or their salts, in which the polycarboxylic acidcomprises at least two carboxyl radicals separated from each other bynot more than two carbon atoms. Suitable dispersants are for exampledescribed in Powdered Detergents, Surfactant science series volume 71,Marcel Dekker, Inc.

Dye Transfer Inhibiting Agents

The detergent compositions of the present invention may also include oneor more dye transfer inhibiting agents. Suitable polymeric dye transferinhibiting agents include, but are not limited to, polyvinylpyrrolidonepolymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidoneand N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles ormixtures thereof. When present in a subject composition, the dyetransfer inhibiting agents may be present at levels from about 0.0001%to about 10%, from about 0.01% to about 5% or even from about 0.1% toabout 3% by weight of the composition.

Fluorescent Whitening Agent

The detergent compositions of the present invention will preferably alsocomprise additional components that may tint articles being cleaned,such as fluorescent whitening agent or optical brighteners. Wherepresent the brightener is preferably at a level of about 0.01% to about0.5%. Any fluorescent whitening agent suitable for use in a laundrydetergent composition may be used in the composition of the presentinvention. The most commonly used fluorescent whitening agents are thosebelonging to the classes of diaminostilbene-sulfonic acid derivatives,diarylpyrazoline derivatives and bisphenyl-distyryl derivatives.Examples of the diaminostilbene-sulfonic acid derivative type offluorescent whitening agents include the sodium salts of:4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulfonate, 4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2,2′-disulfonate,4,4′-bis-(2-anilino-4-(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulfonate,4,4′-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2′-disulfonate andsodium5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate.Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBSavailable from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is thedisodium salt of 4,4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2′-disulfonate. Tinopal CBS is the disodium salt of2,2′-bis-(phenyl-styryl)-disulfonate. Also preferred are fluorescentwhitening agents is the commercially available Parawhite KX, supplied byParamount Minerals and Chemicals, Mumbai, India. Other fluorescerssuitable for use in the invention include the 1-3-diary) pyrazolines andthe 7-alkylaminocoumarins.

Suitable fluorescent brightener levels include lower levels of fromabout 0.01, from 0.05, from about 0.1 or even from about 0.2 wt % toupper levels of 0.5 or even 0.75 wt %.

Soil Release Polymers

The detergent compositions of the present invention may also compriseone or more soil release polymers which aid the removal of soils fromfabrics such as cotton and polyester based fabrics, in particular theremoval of hydrophobic soils from polyester based fabrics. The soilrelease polymers may for example be nonionic or anionic terephthaltebased polymers, polyvinyl caprolactam and related copolymers, vinylgraft copolymers, polyester polyamides see for example Chapter 7 inPowdered Detergents, Surfactant science series volume 71, Marcel Dekker,Inc. Another type of soil release polymers are amphiphilic alkoxylatedgrease cleaning polymers comprising a core structure and a plurality ofalkoxylate groups attached to that core structure. The core structuremay comprise a polyalkylenimine structure or a polyalkanolaminestructure as described in detail in WO 2009/087523 (hereby incorporatedby reference). Furthermore random graft co-polymers are suitable soilrelease polymers. Suitable graft co-polymers are described in moredetail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (herebyincorporated by reference). Other soil release polymers are substitutedpolysaccharide structures especially substituted cellulosic structuressuch as modified cellulose deriviatives such as those described in EP1867808 or WO 2003/040279 (both are hereby incorporated by reference).Suitable cellulosic polymers include cellulose, cellulose ethers,cellulose esters, cellulose amides and mixtures thereof. Suitablecellulosic polymers include anionically modified cellulose, nonionicallymodified cellulose, cationically modified cellulose, zwitterionicallymodified cellulose, and mixtures thereof. Suitable cellulosic polymersinclude methyl cellulose, carboxy methyl cellulose, ethyl cellulose,hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, estercarboxy methyl cellulose, and mixtures thereof.

Anti-Redeposition Agents

The detergent compositions of the present invention may also include oneor more anti-redeposition agents such as carboxymethylcellulose (CMC),polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethyleneand/or polyethyleneglycol (PEG), homopolymers of acrylic acid,copolymers of acrylic acid and maleic acid, and ethoxylatedpolyethyleneimines. The cellulose based polymers described under soilrelease polymers above may also function as anti-redeposition agents.

Rheology Modifiers

The detergent compositions of the present invention may also compriseone or more rheology modifiers, structurants or thickeners, as distinctfrom viscosity reducing agents. The rheology modifiers are selected fromthe group consisting of non-polymeric crystalline, hydroxy-functionalmaterials, polymeric rheology modifiers which impart shear thinningcharacteristics to the aqueous liquid matrix of a liquid detergentcomposition. The rheology and viscosity of the detergent may be modifiedand adjusted by methods known in the art, for example as shown in EP2169040.

Other suitable adjunct materials include, but are not limited to,anti-shrink agents, anti-wrinkling agents, bactericides, binders,carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foamregulators, hydrotropes, perfumes, pigments, sod suppressors, solvents,and structurants for liquid detergents and/or structure elasticizingagents.

Formulation of Detergent Products

The detergent composition of the invention may be in any convenientform, e.g., a bar, a homogenous tablet, a tablet having two or morelayers, a pouch having one or more compartments, a regular or compactpowder, a granule, a paste, a gel, or a regular, compact or concentratedliquid.

Pouches may be configured as single or multicompartments. It may be ofany form, shape and material which is suitable for hold the composition,e.g. without allowing the release of the composition to release of thecomposition from the pouch prior to water contact. The pouch is madefrom water soluble film which encloses an inner volume. Said innervolume may be divided into compartments of the pouch. Preferred filmsare polymeric materials preferably polymers which are formed into a filmor sheet. Preferred polymers, copolymers or derivates thereof areselected polyacrylates, and water soluble acrylate copolymers, methylcellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin,poly methacrylates, most preferably polyvinyl alcohol copolymers and,hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymerin the film for example PVA is at least about 60%. Preferred averagemolecular weight will typically be about 20,000 to about 150,000. Filmsmay also be of blended compositions comprising hydrolytically degradableand water soluble polymer blends such as polylactide and polyvinylalcohol (known under the Trade reference M8630 as sold by MonoSol LLC,Indiana, USA) plus plasticisers like glycerol, ethylene glycerol,propylene glycol, sorbitol and mixtures thereof. The pouches maycomprise a solid laundry cleaning composition or part components and/ora liquid cleaning composition or part components separated by the watersoluble film. The compartment for liquid components may be different incomposition than compartments containing solids: US2009/0011970 A1.

Detergent ingredients may be separated physically from each other bycompartments in water dissolvable pouches or in different layers oftablets. Thereby negative storage interaction between components can beavoided. Different dissolution profiles of each of the compartments mayalso give rise to delayed dissolution of selected components in the washsolution.

A liquid or gel detergent, which is not unit dosed, may be aqueous,typically comprising at least 20% by weight and up to 95% water, such asup to about 70% water, up to about 65% water, up to about 55% water, upto about 45% water, up to about 35% water. Other types of liquids,including without limitation, alkanols, amines, diols, ethers andpolyols may be included in an aqueous liquid or gel. An aqueous liquidor gel detergent may contain from 0-30% organic solvent.

Granular Detergent Formulations

A granular detergent may be formulated as described in WO09/092699,EP1705241, EP1382668, WO07/001262, U.S. Pat. No. 6,472,364, WO04/074419or WO09/102854. Other useful detergent formulations are described inWO09/124162, WO09/124163, WO09/117340, WO09/117341, WO09/117342,WO09/072069, WO09/063355, WO09/132870, WO09/121757, WO09/112296,WO09/112298, WO09/103822, WO09/087033, WO09/050026, WO09/047125,WO09/047126, WO09/047127, WO09/047128, WO09/021784, WO09/010375,WO09/000605, WO09/122125, WO09/095645, WO09/040544, WO09/040545,WO09/024780, WO09/004295, WO09/004294, WO09/121725, WO09/115391,WO09/115392, WO09/074398, WO09/074403, WO09/068501, WO09/065770,WO09/021813, WO09/030632, and WO09/015951.

WO2011025615, WO2011016958, WO2011005803, WO2011005623, WO2011005730,WO2011005844, WO2011005904, WO2011005630, WO2011005830, WO2011005912,WO2011005905, WO2011005910, WO2011005813, WO2010135238, WO2010120863,WO2010108002, WO2010111365, WO2010108000, WO2010107635, WO2010090915,WO2010033976, WO2010033746, WO2010033747, WO2010033897, WO2010033979,WO2010030540, WO2010030541, WO2010030539, WO2010024467, WO2010024469,WO2010024470, WO2010025161, WO2010014395, WO2010044905,

WO2010145887, WO2010142503, WO2010122051, WO2010102861, WO2010099997,WO2010084039, WO2010076292, WO2010069742, WO2010069718, WO2010069957,WO2010057784, WO2010054986, WO2010018043, WO2010003783, WO2010003792,

WO2011023716, WO2010142539, WO2010118959, WO2010115813, WO2010105942,WO2010105961, WO2010105962, WO2010094356, WO2010084203, WO2010078979,WO2010072456, WO2010069905, WO2010076165, WO2010072603, WO2010066486,WO2010066631, WO2010066632, WO2010063689, WO2010060821, WO2010049187,WO2010031607, WO2010000636.

Uses

The present invention is also directed to methods for using thealpha-amylase variants. Accordingly, the present invention relates tomethods for using a variant comprising a) a deletion at two or morepositions corresponding to positions R181, G182, D183 and G184 of themature polypeptide of SEQ ID NO: 1, b) a substitution at one or morepositions corresponding to positions Y198, Y200, L201, Y203, and A204 ofthe mature polypeptide of SEQ ID NO: 1, and c) substitution of themethionine at the position corresponding to position M202 of the maturepolypeptide of SEQ ID NO: 1, wherein the variant has at least 80%, suchas at least 90%, but less than 100% sequence identity with the maturepolypeptide of SEQ ID NO: 1 or 2, and wherein the variant hasalpha-amylase activity.

The alpha-amylase variants of the invention are useful in detergentcompositions, laundry washing, dishwashing and/or cleaning processes atlow temperature as well as hard surface cleaning (ADW, car wash,Industrial surface). The alpha-amylase variants of the invention areparticularly useful in dish wash detergent compositions because they arestable towards oxidation by the bleaching agents and have improvedactivity.

Use in detergents. The polypeptides of the present invention may beadded to and thus become a component of a detergent composition, inparticular a dish wash detergent composition.

The detergent composition of the present invention may be formulated,for example, as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations.

The detergent composition may further be formulated in unit dosage formor in form a soap bar or a laundry bar.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide of the present invention as describedherein.

Methods

-   Amylase expression: the alpha-amylase variants of the present    invention may be expressed as disclosed in WO2010115021 or U.S. Pat.    No. 6,623,948.-   Strain: e.g. B.subtilis, B.licheniformis, carrying the amylase in an    expression cassette either on a plasmid or integrated on the    bacillus chromosome, e.g. in the Pel or Amy locus.-   Media: e.g., LB, TY, Media-16-   Media 16    -   Glycerol—5% w/v    -   Tryptone—0.5% w/v    -   Beef Extract—0.5% w/v    -   Sodium Nitrate—1% w/v    -   Na₂HPO₄—1.7% w/v    -   KH₂PO₄—0.4% w/v    -   NH₄Cl—0.1% w/v    -   NaCl—0.05% w/v    -   Adjust to pH 7 and autoclave.        Autoclaved Separately and Added Just Before Inoculation    -   1.47% CaCl₂—0.4 ml for 100 ml media    -   2.465% MgSO₄.7H₂O—0.4 ml for 100 ml media    -   1.39% FeSO₄—0.04 ml for 100 ml media    -   0.2% Na₂MoO₄.2H₂O—0.04 ml for 100 ml media    -   Vitamin Mix (containing 0.25% Thiamine and 0.25% Ascorbic        Acid)—0.4 ml of Vitamin Mix for 100 ml media    -   Trace Elements (containing 0.5% MnCl₂.4H₂O, 0.2% ZnCl₂ and 0.1%        CuSO₄.5H₂O)—0.04 ml of Trace sol for 100 ml media        Construction of Variants of SEQ ID NO: 2

The wild type amylase was isolated as described in U.S. Pat. No.6,623,948 and cloned into the Pel locus of B. subtilis, so that theup-stream fragment including the upper Pel locus and a down-streamfragment including the lower Pel logi, so that the amylase upontransformation in B. subtilis will integrate in the Pel locus by doublecross-over replacement.

The upper fragment further comprises a triple promoter system (asdescribed in WO 99/43835) consisting of the promoters from Bacilluslicheniformis alpha-amylase gene (amyL), Bacillus amyloliquefaciensalpha-amylase gene (amyQ), and the Bacillus thuringiensis cryIllApromoter including stabilizing sequence controlling the amylaseexpression, and the signal sequence of the B. licheniformis amylasesignal to direct export out of the cells. The down-stream fragmentfurther comprises the cat gene for selection on Chloramphenicolcontaining media.

The double deletion R181*+G182*, and the N195F mutations found in thesequence ID no 2, were introduced by megaprimer mutagenesis method usinga mutagenesis oligo coding for the desired amino acid change, andcloning into the expression cassette as for the reference amylasedescribed above. The sequence was confirmed by DNA sequencing of theamylase gene.

Production and Purification of Amylases

The amylase expressing clones can be fermented in media-16 at 37° C.with 180 rpm for 72 hours and the broths centrifuged at 13131 g for 25minutes to remove the cell mass, and then filtered using a 0.7 micrometer Glass filter GF-F, Whatman using tarsons filtration assembly.

Reference and variant amylases can be purified from the supernatant by24 well plate protein purification method: 3 ml of a 50% slurry of butyltoyopearl resin in milli Q water is to be added into each well of 24well filter plate and the plate subjected to vacuum to pack the columnplate. The resin should be equilibrated by adding 8 mL of equilibrationbuffer (50 mM HEPES, pH 8.0+1 M ammonium sulphate +1 mM CaCl2) and 8 mlof the amylase samples can be then added into the wells of filter plateand incubate on plate mixer at 350 rpm for 8 min. The unbound fractioncan be removed by vacuum and the resin washed by 4 cycles of adding 8 mLof equilibration buffer (50 mM HEPES, pH 8.0+1 M ammonium sulphate +1 mMCaCl2) followed by mixing and incubation and finally removing the washbuffers by vacuum.

The amylase can be eluted by adding elution buffer (50 mM HEPES, pH8.0+1 mM CaCl2), mixed and incubated prior to collecting the amylasesolution in a collection tray by vacuum.

Assays for Measurement of Amylolytic Activity (Alpha-Amylase Activity)

EnzChek Assay

The amylase activity or residual amylase activity can be determined bythe following EnzCheck assay. The substrate is a corn starch derivative,DQTM starch (corn starch BODIPY FL conjugate), which is corn starchlabelled with BODIPYO FL(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid) dye to such a degree that the fluorescence is quenched. One vialcontaining approx. 1 mg lyophilized substrate is dissolved in 100 μL 50mM sodium acetate pH 4.0. The vial is vortexed for 20 seconds and leftat room temperature, in the dark, with occasional mixing untildissolved. Then 950 μL 10 mM sodium acetate, 0.01% (w/V) TRITON-X-100®((polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether(C14H22O(C2H4O)n (n=9-10)), pH 5.0 is added, vortexed thoroughly andstored at room temperature, in the dark until ready to use. From 1 mL ofthis solution, the substrate working solution was prepared by mixingwith 5 mL 50 mM HEPES, 0.01% (w/V) TRITON-X-100®, 1 mM CaCl₂), pH 7.0.

The enzyme comprising detergent is diluted to a concentration of 15 ngenzyme protein/ml (6826.7 times dilution) in 50 mM HEPES, 0.01%TRITON-X-100®, 1 mM CaCl₂), pH 7.0.

For the assay, 25 μL of the substrate working solution is mixed for 10second with 25 μL of the diluted enzyme in a black 384 well microtiterplate. The fluorescence intensity is measured (ex-citation: 485 nm,emission: 555 nm) once every second minute for 30 minutes in each wellat 25° C. and the Vmax is calculated as the slope of the plot offluorescence intensity against time. The plot should be linear and theresidual activity assay has to been adjusted so that the dilutedreference enzyme solution is within the linear range of the activityassay.

In a few instances there is a significant interference from thedetergent without amylase on the assay. In such cases alternativeamylase assays can be used. Interference from a detergent on an amylaseassay can be tested by adding a known amount of amylase to the detergentat two levels and then measure the activity of the two samples. If thedifference in the measured activities corresponds to the differences inthe levels between the added amylases, the assay can be used todetermine the residual activity of the amylase after storage.

PNP-G7 Assay

The alpha-amylase activity may be determined by a method employing thePNP-G7 substrate. PNP-G7 which is an abbreviation for4,6-ethylidene(G7)-p-nitrophenyl(G1)-α,D-maltoheptaoside, a blockedoligosaccharide which can be cleaved by an endo-amylase, such as analpha-amylase. Following the cleavage, the alpha-Glucosidase included inthe kit digest the hydrolysed substrate further to liberate a free PNPmolecule which has a yellow color and thus can be measured by visiblespectophometry at λ=405 nm (400-420 nm.). Kits containing PNP-G7substrate and alpha-Glucosidase are manufactured by Roche/Hitachi (cat.No. 11876473).

Reagents:

The G7-PNP substrate from this kit contains 22 mM 4,6-ethylidene-G7-PNPand 52.4 mM HEPES (2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonicacid), pH 7.0).

The alpha-Glucosidase reagent comprises 52.4 mM HEPES, 87 mM NaCl, 12.6mM MgCl2, 0.075 mM CaCl2, >4 kU/L alpha-glucosidase).

The substrate working solution is made by mixing 1 mL of thealpha-Glucosidase reagent with 0.2 mL of the G7-PNP substrate. Thissubstrate working solution is made immediately before use.

Dilution buffer: 50 mM EPPS, 0.01% (w/v) TRITON-X-100® (polyethyleneglycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether (C14H22O(C2H4O)n(n=9-10))), 1 mM CaCl₂), pH7.0.

Procedure:

The amylase sample to be analysed was diluted in dilution buffer toensure the pH in the diluted sample is 7. The assay was performed bytransferring 20 μl diluted enzyme samples to 96 well microtiter plateand adding 80 μl substrate working solution. The solution was mixed andpre-incubated 1 minute at room temperature and absorption is measuredevery 20 sec. over 5 minutes at OD 405 nm.

The slope (absorbance per minute) of the time dependent absorption-curveis directly proportional to the specific activity (activity per mgenzyme) of the alpha-amylase in question under the given set ofconditions. The amylase sample should be diluted to a level where theslope is below 0.4 absorbance units per minute.

Determination of Percentage Point (Pp)

The percentage point (pp) improvement in residual activity (stability)of the variant relative to the parent is calculated as the differencebetween the residual activity of the variant and the residual activityof the parent, i.e. the residual activity of the variant minus theresidual activity of the parent.

Amylazyme Activity Assay:

The alpha-amylase activity may also be determined by a method using theAmylazyme substrate (from Megazyme, Ireland). An Amylazyme tabletincludes interlinked amylose polymers that are in the form of globularmicrospheres that are insoluble in water. A blue dye is covalently boundto these microspheres. The interlinked amylose polymers in themicrosphere are degraded at a speed that is proportional to thealpha-amylase activity. When the alpha-amylase degrades the amylosepolymers, the released blue dye is water soluble and concentration ofdye can be determined by measuring absorbance at 650 nm. Theconcentration of blue is proportional to the alpha-amylase activity inthe sample.

The amylase sample to be analysed is diluted in activity buffer with thedesired pH. One substrate tablet is suspended in 5 mL activity bufferand mixed on magnetic stirrer. During mixing of substrate transfer 150μl to microtiter plate (MTP). Add 30 μl diluted amylase sample to 150 μlsubstrate and mix. Incubate for 15 minutes at 37° C. The reaction isstopped by adding 30 μl 1 M NaOH and mix. Centrifuge MTP for 5 minutesat 4000×g. Transfer 100 μl to new MTP and measure absorbance at 620 nm.

The amylase sample should be diluted so that the absorbance at 650 nm isbetween 0 and 2.2, and is within the linear range of the activity assay.

Phadebas Activity Assay

The alpha-amylase activity may also be determined by a method using thePhadebas substrate (from for example Magle Life Sciences, Lund, Sweden).A Phadebas tablet includes interlinked starch polymers that are in theform of globular microspheres which are insoluble in water. A blue dyeis covalently bound to these microspheres. The interlinked starchpolymers in the microsphere are degraded at a speed that is proportionalto the alpha-amylase activity. When the alpha-amylase degrades thestarch polymers, the released blue dye is water soluble andconcentration of dye can be determined by measuring absorbance at 650nm. The concentration of blue is proportional to the alpha-amylaseactivity in the sample.

The amylase sample to be analysed is diluted in activity buffer with thedesired pH. One substrate tablet is suspended in 5 mL activity bufferand mixed on magnetic stirrer. During mixing of substrate transfer 150μl to microtiter plate (MTP). Add 30 μl diluted amylase sample to 150 μlsubstrate and mix. Incubate for 15 minutes at 37° C. The reaction isstopped by adding 30 μl 1 M NaOH and mix. Centrifuge MTP for 5 minutesat 4000×g. Transfer 100 μl to new MTP and measure absorbance at 620 nm.

The amylase sample should be diluted so that the absorbance at 650 nm isbetween 0 and 2.2, and is within the linear range of the activity assay.

Reducing Sugar Activity Assay

The alpha-amylase activity may also be determined by reducing sugarassay with for example corn starch substrate. The number of reducingends formed by the alpha-amylase hydrolysing the alpha-1,4-glycosidiclinkages in starch is determined by reaction with p-Hydroxybenzoic acidhydrazide (PHBAH). After reaction with PHBAH the number of reducing endscan be measured by absorbance at 405 nm and the concentration ofreducing ends is proportional to the alpha-amylase activity in thesample.

The corns starch substrate (3 mg/ml) is solubilised by cooking for 5minutes in milliQ water and cooled down before assay. For the stopsolution prepare a Ka-Na-tartrate/NaOH solution (K—Na-tartrate (Merck8087) 50 g/l, NaOH 20 g/l) and prepare freshly the stop solution byadding p-Hydroxybenzoic acid hydrazide (PHBAH, Sigma H9882) toKa-Na-tartrate/NaOH solution to 15 mg/ml.

In PCR-MTP 50 μl activity buffer is mixed with 50 μl substrate. Add 50μl diluted enzyme and mix. Incubate at the desired temperature in PCRmachine for 5 minutes. Reaction is stopped by adding 75 μl stop solution(Ka-Na-tartrate/NaOH/PHBAH). Incubate in PCR machine for 10 minutes at95° C. Transfer 150 μl to new MTP and measure absorbance at 405 nm.

The amylase sample should be diluted so that the absorbance at 405 nm isbetween 0 and 2.2, and is within the linear range of the activity assay.

Wash Performance of Alpha-Amylases using Automatic Mechanical StressAssay

In order to assess the wash performance of the alpha-amylases in adetergent base composition, washing experiments may be performed usingAutomatic Mechanical Stress Assay (AMSA). With the AMSA test the washperformance of a large quantity of small volume enzyme-detergentsolutions can be examined. The AMSA plate has a number of slots for testsolutions and a lid firmly squeezing the textile swatch to be washedagainst all the slot openings. During the washing time, the plate, testsolutions, textile and lid are vigorously shaken to bring the testsolution in contact with the textile and apply mechanical stress in aregular, periodic oscillating manner. For further description see WO02/42740, especially the paragraph “Special method embodiments” at page23-24.

General Wash Performance Description

A test solution comprising water (6° dH), 0.79 g/L detergent, e.g. modeldetergent J as described below, and the enzyme of the invention atconcentration of 0 or 0.2 mg enzyme protein/L, is prepared. Fabricsstained with starch (CS-28 from Center For Test materials BV, P.O. Box120, 3133 KT, Vlaardingen, The Netherlands) is added and washed for 20minutes at 15° C. and 30° C., or alternatively 20 minutes at 15° C. and40° C. as specified in the examples. After thorough rinse under runningtap water and drying in the dark, the light intensity values of thestained fabrics are subsequently measured as a measure for washperformance. The test with 0 mg enzyme protein/L is used as a blank andcorresponds to the contribution from the detergent. Preferablymechanical action is applied during the wash step, e.g. in the form ofshaking, rotating or stirring the wash solution with the fabrics. TheAMSA wash performance experiments were conducted under the experimentalconditions specified below:

TABLE A Experimental condition Detergent Liquid Model detergent J (seeTable B) Detergent dosage 0.79 g/L Test solution volume 160 micro L pHAs is Wash time 20 minutes Temperature 15° C. or 30° C. Water hardness6°dH Enzyme concentration in test 0.2 mg enzyme protein/L Test materialCS-28 (Rice starch cotton)

TABLE B Model detergent J Content of compound % active componentCompound (% w/w) (% w/w) LAS 5.15 5.00 AS 5.00 4.50 AEOS 14.18 10.00Coco fatty acid 1.00 1.00 AEO 5.00 5.00 MEA 0.30 0.30 MPG 3.00 3.00Ethanol 1.50 1.35 DTPA (as Na5 salt) 0.25 0.10 Sodium citrate 4.00 4.00Sodium formate 1.00 1.00 Sodium hydroxide 0.66 0.66 H₂O, ion exchanged58.95 58.95

Water hardness was adjusted to 6° dH by addition of CaCl₂, MgCl₂, andNaHCO₃ (Ca²⁺:Mg²⁺:HCO³⁻=2:1:4.5) to the test system. After washing thetextiles were flushed in tap water and dried.

TABLE C Experimental condition Detergent Liquid Model detergent A (seeTable D) Detergent dosage 3.33 g/L Test solution volume 160 micro L pHAs is Wash time 20 minutes Temperature 15° C. or 40° C. Water hardness15°dH Enzyme concentration in test 0.2 mg enzyme protein/L Test materialCS-28 (Rice starch cotton)

TABLE D Model detergent A Content of compound % active componentCompound (% w/w) (% w/w) LAS 12.00 11.60 AEOS, SLES 17.63 4.90 Soy fattyacid 2.75 2.48 Coco fatty acid 2.75 2.80 AEO 11.00 11.00 Sodiumhydroxide 1.75 1.80 Ethanol/Propan-2-ol 3.00 2.70/0.30 MPG 6.00 6.00Glycerol 1.71 1.70 TEA 3.33 3.30 Sodium formate 1.00 1.00 Sodium citrate2.00 2.00 DTMPA 0.48 0.20 PCA 0.46 0.18 Phenoxy ethanol 0.50 0.50 H₂O,ion exchanged 33.64 33.64

Water hardness was adjusted to 15° dH by addition of CaCl₂, MgCl₂, andNaHCO₃ (Ca²⁺:Mg²⁺:HCO³⁻=4:1:7.5) to the test system. After washing thetextiles were flushed in tap water and dried.

TABLE E Experimental condition Detergent Powder Model detergent X (seeTable F) Detergent dosage 1.75 g/L Test solution volume 160 micro L pHAs is Wash time 20 minutes Temperature 15° C. or 30° C. Water hardness12°dH Enzyme concentration in test 0.2 mg enzyme protein/L Test materialCS-28 (Rice starch cotton)

TABLE F Model detergent X Content of compound % active componentCompound (% w/w) (% w/w) LAS 16.50 15.00 AEO* 2.00 2.00 Sodium carbonate20.00 20.00 Sodium (di)silicate 12.00 9.90 Zeolite A 15.00 12.00 Sodiumsulfate 33.50 33.50 PCA 1.00 1.00 *Model detergent X is mixed withoutAEO. AEO is added separately before wash.Water hardness was adjusted to 12° dH by addition of CaCl₂, MgCl₂, andNaHCO₃ (Ca²⁺:Mg²⁺:HCO₃ ⁻=2:1:4.5) to the test system. After washing thetextiles were flushed in tap water and dried.

Model detergents 1 and 2 Model 1 Model 2 Component % w/w % w/w LAS 12 12AEOS 4.9 4.9 Soap (cocoa) 2.75 2.75 Soap (soya) 2.75 2.75 AEO N25-7 1111 NaOH 1.75 1.75 Ethanol 3 3 MPG 6 6 Glycerol 1.7 1.7 TEA 3.3 3.3Sodium formate 1 1 Sodium citrate 2 2 HEDP 0 0.5 PCA (Sokalan CP-5) 0.180.18 Ion exchanged water 34.2 34.2 DTMPA 0.2 0

The wash performance is measured as the brightness expressed as theintensity of the light reflected from the sample when illuminated withwhite light. When the sample is stained the intensity of the reflectedlight is lower, than that of a clean sample. Therefore the intensity ofthe reflected light can be used to measure wash performance.

Color measurements are made with a professional flatbed scanner (KodakiQsmart, Kodak) used to capture an image of the washed textile.

To extract a value for the light intensity from the scanned images,24-bit pixel values from the image are converted into values for red,green and blue (rgb). The intensity value (Int) is calculated by addingthe rgb values together as vectors and then taking the length of theresulting vector:Int=√{square root over (r ² +g ² +b ²)}Textile:

Textile sample CS-28 (rice starch on cotton) is obtained from Center ForTest materials BV, P.O. Box 120, 3133 KT Vlaardingen, the Netherlands.

Automatic Mechanical Stress Assay (AMSA) for Automatic Dish Wash

Washing experiments are performed in order to assess the washperformance of selected alpha-amylase variants in dishwash detergentcompositions. The alpha-amylase variants of the present application maybe tested using the Automatic Mechanical Stress Assay (AMSA). With theAMSA, the wash performance of many small volume enzyme-detergentsolutions can be examined. The AMSA plate has a number of slots for testsolutions and a lid that firmly squeezes the melamine tile to be washedagainst the slot openings. During the wash, the plate, test solutions,melamine tile and lid are vigorously shaken to bring the test solutionin contact with the soiled melamine tile and apply mechanical stress ina regular, periodic oscillating manner. For further description see WO02/42740 especially the paragraph “Special method embodiments” at page23-24.

The experiment may be conducted under the experimental conditions asspecified in the table(s) below:

ADW model detergent with MGDA MGDA (40%) 30% Sodium carbonate 20% Sodiumpercarbonate 10% Sodium disilicate 5% TAED 5% Sokalan CP5 (39.5%) 10%Surfac 23-6.5 (100%) 5% Sodium Sulfate 15% Detergent dosage 3.33 g/LTest solution volume 160 micro L pH As is Wash time 20 minutesTemperature 50° C. Water hardness 17°dH Enzyme concentration in testsolution 0.925, 1.85, 5.55, 11 mg enzyme protein/liter Test materialmelamine tiles with starch such as DM-77 and DM-78

ADW model detergent with STPP STPP 50% Sodium carbonate 20% Sodiumpercarbonate 10% Sodium disilicate 5% TAED 2% Sokalan CP5 (39.5%) 5%Surfac 23-6.5 (100%) 2% Phosphonate 6% Detergent dosage 3.33 g/L Testsolution volume 160 micro L pH As is Wash time 20 minutes Temperature50° C. Water hardness 17°dH Enzyme concentration in test solution 0.925,1.85, 5.55, 11 mg enzyme protein/liter Test material melamine tiles withstarch such as DM-77 and DM-78

Water hardness is adjusted to 17° dH by addition of CaCl₂, MgCl₂, andNaHCO₃ (Ca²⁺:Mg²+=4:1:10) to the test system. After washing the melaminetiles were flushed in tap water and dried.

The performance of the enzyme variant is measured as the brightness ofthe color of the melamine tile washed with that specific alpha-amylase.Brightness can also be expressed as the intensity of the light reflectedfrom the sample when illuminated with white light. When the sample isstained the intensity of the reflected light is lower, than that of aclean sample. Therefore the intensity of the reflected light can be usedto measure wash performance of a protease.

Color measurements are made with a professional flatbed scanner (KodakiQsmart, Kodak, Midtager 29, DK-2605 Brøndby, Denmark), which is used tocapture an image of the washed melamine tiles.

To extract a value for the light intensity from the scanned images, aspecial designed software application is used (Novozymes Colour VectorAnalyzer). The program retrieves the 24 bit pixel values from the imageand converts them into values for red, green and blue (RGB). Theintensity value (Int) is calculated by adding the RGB values together asvectors and then taking the length of the resulting vector:Int=√{square root over (r ² +g ² +b ²)}.Textiles: Standard melamine tiles with starch such as DM-77 and DM-78may be obtained from Center For Testmaterials BV, P.O. Box 120, 3133 KTVlaardingen, the Netherlands.Terg-O-Tometer (TOM) Wash Assay

The Tergo-To-Meter (TOM) is a medium scale model wash system that can beapplied to test 12 different wash conditions simultaneously. A TOM isbasically a large temperature controlled water bath with up to 12 openmetal beakers submerged into it. Each beaker constitutes one small toploader style washing machine and during an experiment, each of them willcomprise a solution of a specific detergent/enzyme system and the soiledand unsoiled fabrics its performance is tested on. Mechanical stress isachieved by a rotating stirring arm, which stirs the liquid within eachbeaker. Because the TOM beakers have no lid, it is possible to withdrawsamples during a TOM experiment and assay for information on-line duringwash.

The TOM model wash system is mainly used in medium scale testing ofdetergents and enzymes at US or LA/AP wash conditions. In a TOMexperiment, factors such as the ballast to soil ratio and the fabric towash liquor ratio can be varied. Therefore, the TOM provides the linkbetween small scale experiments, such as AMSA and mini-wash, and themore time consuming full scale experiments in top loader washingmachines.

Equipment: The water bath with 12 steel beakers and 1 rotating arm perbeaker with capacity of 500 or 1200 mL of detergent solution.Temperature ranges from 5 to 80° C. The water bath has to be filled upwith deionised water. Rotational speed can be set up to 70 to 120rpm/min.

TOM Wash Performance

Water hardness is adjusted to the strength described below by additionof CaCl₂, MgCl₂ and NAHCO₃. Wash solutions are prepared with desiredamount of detergent, temperature and water hardness in a bucket asdescribed below. Detergent is dissolved during magnet stirring for 10min.

Temperature and rotation (rpm) in the water bath in the Terg-O-Tometeris set according to the settings below. When temperature is adjustedaccording to settings (tolerance is +/−0.5° C.) wash solution is addedto TOM beaker according to the amount described below.

Agitation in the beaker is at 120 rpm. 2 rice starch swatches (CS-28)and soil ballast are added to each of the beakers and wash carried outaccording to time stated below. Swatches are rinsed in cold tap waterfor 5 min. The swatches are left to dry in the dark overnight.

Textile: Textile sample CS-28 (rice starch on cotton) is obtained fromCenter for Test materials BV, P.O. Box 120, 3133 KT Vlaardingen, theNetherlands.

Soil ballast: Soil ballast Rice starch on cotton/polyester (EMPA 162) isobtained from Center for Test materials BV, P.O. Box 120, 3133 KTVlaardingen, the Netherlands. Bistro gravy (063KC), Frij Chocolatemilkshake, Heinz spaghetti (113KC), Herseys double chocolate is obtainedfrom Warwick Equest Ltd, Unit 55, Consett Business Park, Consett, CountyDurham, DH8 6BN UK

Experimental conditions TOM Northern European America (US) (EU)conditions conditions Detergent dosage 5.77 g/L 0.78 g/L (liquiddetergent) (liquid detergent) Water hardness 15°dH 6°dH (Ca²⁺:Mg²⁺:HCO₃⁻ = (Ca²⁺:Mg²⁺:HCO₃ ⁻ = 4:1:7.5) 2:1:4.5) Enzyme concentration in 0.25mg enzyme 0.08 mg enzyme wash solution protein/L protein/L Test solutionvolume 500 ml 800 ml Wash time  30 minutes  18 minutes Rotation 120 rpmpH as is Temperature  15° C.

Detergents and test materials may be as follows: Laundry liquiddetergent May be as described below Test material CS-28 (Rice starch oncotton) Soil ballast Rice starch on polyester/cotton (EMPA 162), Bistrogravy (063KC), Frij Chocolate milkshake, Heinz spaghetti (113KC),Herseys double chocolate (2 swatches of each)

The wash performance is measured as the brightness of the color of thetextile washed, expressed in remission values. Remission measurementsare made using a Macbeth 7000 Color Eye spectrophotometer. Each of thedry swatches is to be measured. As there is a risk of interference fromthe back-ground, the swatches are placed on top of 4 layers of fabricduring the measurement of the remission. The remission is measured at460 nm. The UV filter is not included. An average result for remissionfor the swatches is calculated.

EXAMPLES Example 1: Identification of Variants Having Alpha-AmylaseActivity

A library of multiple substitutions in the loop spanning from aminoacids 198-204 when using SEQ ID NO: 1 for numbering was designed, theamylase DNAs generated by Slonomics Technology using SEQ ID NO: 2 asstarting point, and the amylase expression cassette was transformed intoB.subtilis to provide a library. Clones expressing significant amount ofamylase activity using the Enzcheck substrate were isolated and thecombination of substitutions identified by sequencing. Table G shows theamino acid sequence of amino acids 198-204 for variants of SEQ. ID NO 2(using SEQ ID NO: 1 for numbering).

TABLE G amino acid sequence of amino acids 198-204using SEQ ID NO: 1 for numbering YDYLLFA YDWLLYA YDWLLFA YDWLLPA YDYLLIAYDYLLNA YDYLLPA YDYQLYA YDPLLYA YDYLLTA YDYLLWA YDNLLYA YDQLLLA YDQLLYAYDWLLWA YDYLLVA YDQLLPA YDQLLWA YDYLLHA YDYLLLA YDYLLSA YDYPLYA YDYQPAAYDELLYA YDKLLPA YDQLLNA YDYELYA YDYHLYA YDYLLDA YDYLPRA YDYQLLA YDYQLPAYDYQLQA YDDLLYA YDKLLYA YDWLLHA YDWLLTA YDWLPSA YDWQLYA YDYGLYA YDYLLEAYDYLLQA YDYPLPA YDYRLYA YDIELSA YDQLLIA YDQLLSA YDWLGYA YDWLLAA YDWQLHAYDWWLPA YDYELLA YDYLFTA YDYLLKA YDYLYSA YDYQLFA YDYQYYA YDYTLYA YDYYLYAYDIELWA YDIELYA YDNLLNA YDNLLPA YDPLLHA YDQLLVA YDQLPYA YDWLLRA YDWLWYAYDWWLGA YDYHLIA YDYQHYA YDYQLGA YDYQLIA YDYWLPA YDELLWA YDHLLNA YDIELLAYDIELNA YDIELRA YDILLYA YDKLLWA YDLPLYA YDNLLLA YDPLLAA YDPLLPA YDQHLPAYDQLLEA YDQLLQA YDQLNYA YDQLPFA YDQLPNA YDQLPRA YDTLLLA YDTLLYA YDVLLYAYDWLLKA YDWLLLA YDWLLNA YDWLLQA YDWLLVA YDWLPPA YDWLPTA YDWPWYA YDWWLWAYDYHLFA YDYHPSA YDYLLNT YDYLLYT YDYLPFA YDYLPSA YDYLVSA YDYLYPA YDYLYRAYDYPLFA YDYPLSA YDYPLTA YDYPQYA YDYQLTA YDYQLWA YDYQNYA YDYQPRA YDYQSHAYDYREYA YDYRLPA YDYRNSA YDYRPRA YDYTQYA YDYVLYA YDYWLSA YDDLLSA YDELLDAYDELLPA YDELLTA YDEQLEA YDEQLYA YDGLPHA YDIELFA YDIELKA YDIELPA YDKLLNAYDKPPSA YDLLLFA YDLPLLA YDNLLKA YDPLKFA YDPLLEA YDPLLFA YDPLLKA YDPLLWAYDPPLPA YDPPLYA YDPTLPA YDPTQYA YDQELPA YDQLDHA YDQLEYA YDQLLDA YDQLLFAYDQLLNT YDQLLYS YDQLLYT YDQLPSA YDQLWYA YDQQLVA YDTLLWA YDTPLFA YDTPLYAYDWELYA YDWHLYA YDWLHSA YDWLLEA YDWLLIA YDWLLSA YDWLNYA YDWLPFA YDWLPRAYDWLQPA YDWLQYA YDWLWGA YDWPLHA YDWQLRA YDWQLTA YDWSLPA YDWSLYA YDWWLYAYDYELEA YDYELNA YDYGLAA YDYHEWA YDYHLPA YDYHLSA YDYHQYA YDYHTSA YDYLFQAYDYLHLA YDYLIEA YDYLLFT YDYLLRA YDYLLYG YDYLPDA YDYLPQA YDYLPWA YDYLQEAYDYPGYA YDYPHSA YDYPLLA YDYPLNA YDYPNYA YDYPSRA YDYPWYA YDYQEYA YDYQLAAYDYQLKA YDYQLPT YDYQPTA YDYQPYA YDYQQYA YDYQWYA YDYRLFA YDYRPSA YDYRTFAYDYRTSA YDYRTYA YDYSLYA YDYSVYA YDYTPRA YDYWLFA YDYWLGA YDYWLWA YDYWLYAYDDLLLA YDDLLNA YDDLPFA YDEHLHA YDELLFA YDELLSA YDELQIA YDEWPYA YDGLLSAYDHLLYA YDIELHA YDIELKT YDIELTA YDIEVSA YDIPLYA YDIRGYA YDIRNYA YDIRTKAYDIWLYA YDKLPHA YDKLQYA YDKPLSA YDLLLVA YDNHLPA YDNHLYA YDNLGYA YDNLLIAYDNLLVA YDNLLWA YDNLPRA YDNQLYA YDNRLYA YDPHRHA YDPLHVA YDPLLDA YDPLLLAYDPLQYA YDPPQFA YDPQLIA YDQELYA YDQLFSA YDQLKYA YDQLLAA YDQLLHA YDQLNNAYDQLPAA YDQLPPA YDQLQNA YDQLWGT YDQLWPA YDQLYPA YDQNLYA YDQPLPA YDQQLQAYDQTLYA YDQWLHA YDQWLTA YDRLLPA YDSELYA YDTLIRA YDTLLKA YDTLLNA YDTPLNAYDTPLPA YDTPQIA YDTRLYA YDTSLPA YDTTLPA YDTWKYA YDVLLPA YDVLNTA YDWELIAYDWHLPA YDWHQYA YDWHSHA YDWHTQA YDWLHHA YDWLHYA YDWLNWA YDWLPAA YDWLPGAYDWLPIA YDWLQVA YDWLTPA YDWLTQA YDWNLSA YDWNWYA YDWPGYA YDWPLIA YDWPLVAYDWPLYA YDWPTYA YDWQLIA YDWQLLA YDWQLNA YDWWLDA YDYDLYA YDYEKYA YDYELIAYDYELPA YDYELTA YDYELWA YDYGWPA YDYGWYA YDYHENA YDYHHEA YDYHIEA YDYHLQAYDYHPRA YDYHTIA YDYHTPA YDYHTYA YDYLFPA YDYLHWA YDYLIRA YDYLNDA YDYLNPAYDYLNQA YDYLPEA YDYLPHA YDYLPIA YDYLPLA YDYLPTA YDYLQNA YDYLQRA YDYLRQAYDYLWGA YDYLWLA YDYLWPA YDYPEPA YDYPIDA YDYPIRA YDYPLAA YDYPLQA YDYPPRAYDYPQHA YDYPSYA YDYPTAA YDYPTDA YDYQHRA YDYQHSA YDYQIYA YDYQLEA YDYQLLTYDYQNPA YDYQQNA YDYQQSA YDYQTVA YDYQWPA YDYQYRA YDYRHTA YDYRLNA YDYRQYAYDYRRSA YDYRSDA YDYSGYA YDYSNYA YDYSTFA YDYTEYA YDYTLSA YDYTQSA YDYWLEAYDYWLGT YDYWLHA YDYWLLA YDYWLTA YDYWPEA YDYWPRA YDYYLRA

Example 2: Test of Amylase Activity

The generated variants were tested in the above-described pNP-G7 assayin order to determine the amylase activity after binding to an antibody.

The antibody was diluted in Phosphate buffered saline (PBS) (0.010 MPhosphate buffer pH7.4, 0.0027M KCl, 0.14M NaCl) buffer to aconcentration of 10 μg/ml. A maxisorp microtiter plate was coated withantibody by adding 100 μl diluted antibody (10 μg/ml) to each well andincubated for 1 h at room temperature (RT) followed by mixing at 800rpm. After incubation the microtiter plate was washed (using Bio-TekELx405 ELISA washer) with 3×200 μl Phosphate buffered saline with 0.05%TWEEN® (PBST) (0.010 M Phosphate buffer pH7.4, 0.0027M KCl, 0.14M NaCl,0.05%) buffer.

Microtiter plates with amylase variants culture broths were spun downand supernatants transferred to new microtiter plates and diluted 4× inPBST buffer. 100 μl diluted supernatant was transferred to the antibodycoated maxisorp microtiter plate and incubated for 1 h at RT and mixingat 800 rpm. After incubation microtiter plates were washed in PBSTbuffer (3×200 μl, ELISA washer).

The amylase activity of the amylase variants bound to the antibody wasmeasured by addition of 100 μl pNP-G7 substrate to all wells and mixedfor 1 minute before absorbance at 405 nm was measured. The slope(absorbance per minute) was determined and only the linear range ofcurve was used.

Results were compared to a reference sample and samples with higheractivity than the reference sample indicates an improved specificactivity on the pNP-G7 substrate under the tested conditions.

TABLE H Results of specific activity assay Variants of SEQ ID NO: 2 (SEQID NO: 1 Average Improvement numbering) Activity factor (IF) SEQ ID NO:2 + M202L (reference) 18.22 1 SEQ ID NO: 2 + Y200Q + M202L + A204S 18.911.06 SEQ ID NO: 2 + M202L + Y203L 22.80 1.22 SEQ ID NO: 2 + Y200H +M202L + Y203N 26.08 1.38

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

The invention claimed is:
 1. An alpha-amylase variant comprising: a) adeletion at two or more positions corresponding to positions R181, G182,D183 and G184 of the mature polypeptide of SEQ ID NO: 1, b) asubstitution at one or more positions corresponding to positions Y198,Y200, L201, Y203 and A204 of the mature polypeptide of SEQ ID NO: 1, andc) a substitution of the methionine at the position corresponding toposition M202 of the mature polypeptide of SEQ ID NO: 1, wherein thevariant has at least 90%, but less than 100% sequence identity with themature polypeptide of SEQ ID NO: 1, and wherein the variant hasalpha-amylase activity.
 2. The variant of claim 1, wherein thesubstitution b) is selected from one or more of Y198F,L,H,Q,Y200F,L,S,C,W,P,H,Q,R,I,M,T,N,K,V,A,D,E,G,L201F,S,Y,C,W,P,H,Q,R,I,M,T,N,K,V,A,D,E,G,Y203F,L,S,C,W,P,H,Q,R,I,M,T,N,K,V,A,D,E,G, and A204I,M,T,S,R,V,G.
 3. Thevariant of claim 1, wherein the substitution c) is any ofM202F,L,S,Y,C,W,P,H,Q,R,I,T,N,K,V,A,D,E,G.
 4. The variant of claim 1,wherein the substitutions b) and/or c) is selected from one or more of:Y200H, Y200Q, M202L, Y203N, Y203L, A204S, Y200H+M202L, Y200Q+M202L,M202L+Y203N, M202L+Y203L, M202L+A204S, Y200H+M202L+Y203N,Y200H+M202L+Y203L, Y200H+M202L+A204S, Y200Q+M202L+Y203N,Y200Q+M202L+Y203L, Y200Q+M202L+A204S, Y200H+M202L+Y203N+A204S,Y200H+M202L+Y203L+A204S, Y200Q+M202L+Y203N+A204S, andY200Q+M202L+Y203L+A204S.
 5. The variant of claim 1, the variantcomprises an alteration selected from the group consisting of:R181*+G182*+Y200H+M202L, R181*+G182*+Y200Q+M202L,R181*+G182*+M202L+Y203N, R181*+G182*+M202L+Y203L,R181*+G182*+M202L+A204S, R181*+G182*+Y200H+M202L+Y203N,R181*+G182*+Y200H+M202L+Y203L, R181*+G182*+Y200H+M202L+A204S,R181*+G182*+Y200Q+M202L+Y203N, R181*+G182*+Y200Q+M202L+Y203L,R181*+G182*+Y200Q+M202L+A204S, R181*+G182*+Y200H+M202L+Y203N+A204S,R181*+G182*+Y200H+M202L+Y203L+A204S,R181*+G182*+Y200Q+M202L+Y203N+A204S,R181*+G182*+Y200Q+M202L+Y203L+A204S, R181*+D183*+Y200H+M202L,R181*+D183*+Y200Q+M202L, R181*+D183*+M202L+Y203N,R181*+D183*+M202L+Y203L, R181*+D183*+M202L+A204S,R181*+D183*+Y200H+M202L+Y203N, R181*+D183*+Y200H+M202L+Y203L,R181*+D183*+Y200H+M202L+A204S, R181*+D183*+Y200Q+M202L+Y203N,R181*+D183*+Y200Q+M202L+Y203L, R181*+D183*+Y200Q+M202L+A204S,R181*+D183*+Y200H+M202L+Y203N+A204S,R181*+D183*+Y200H+M202L+Y203L+A204S,R181*+D183*+Y200Q+M202L+Y203N+A204S,R181*+D183*+Y200Q+M202L+Y203L+A204S, G182*+G184*+Y200H+M202L,G182*+G184*+Y200Q+M202L, G182*+G184*+M202L+Y203N,G182*+G184*+M202L+Y203L, G182*+G184*+M202L+A204S,G182*+G184*+Y200H+M202L+Y203N, G182*+G184*+Y200H+M202L+Y203L,G182*+G184*+Y200H+M202L+A204S, G182*+G184*+Y200Q+M202L+Y203N,G182*+G184*+Y200Q+M202L+Y203L, G182*+G184*+Y200Q+M202L+A204S,G182*+G184*+Y200H+M202L+Y203N+A204S,G182*+G184*+Y200H+M202L+Y203L+A204S,G182*+G184*+Y200Q+M202L+Y203N+A204S,G182*+G184*+Y200Q+M202L+Y203L+A204S, D183*+G184*+Y200H+M202L,D183*+G184*+Y200Q+M202L, D183*+G184*+M202L+Y203N,D183*+G184*+M202L+Y203L, D183*+G184*+M202L+A204S,D183*+G184*+Y200H+M202L+Y203N, D183*+G184*+Y200H+M202L+Y203L,D183*+G184*+Y200H+M202L+A204S, D183*+G184*+Y200Q+M202L+Y203N,D183*+G184*+Y200Q+M202L+Y203L, D183*+G184*+Y200Q+M202L+A204S,D183*+G184*+Y200H+M202L+Y203N+A204S,D183*+G184*+Y200H+M202L+Y203L+A204S,D183*+G184*+Y200Q+M202L+Y203N+A204S, D183*+G184*+Y200Q+M202L+Y203L+A204SN195F+M202L+R181*+G182*, N195F+M202L+R181*+D183*,N195F+M202L+R181*+G184*, N195F+M202L+G182*+D183*,N195F+M202L+D183*+G184*, N195Y+M202L+R181*+G182*,N195Y+M202L+R181*+D183*, N195Y+M202L+R181*+G184*,N195Y+M202L+G182*+D183*, N195Y+M202L+D183*+G184*R181*+G182*+N195F+Y200H+M202L+Y203N,R181*+G182*+N195F+Y200Q+M202L+A204S, R181*+G182*+N195F+M202L+Y203L,G182*+G184*+N195F+Y200H+M202L+Y203N,G182*+G184*+N195F+Y200Q+M202L+A204S, G182*+G184*+N195F+M202L+Y203L,G182*+D 183*+N195F+Y200H+M202L+Y203N,G182*+D183*+N195F+Y200Q+M202L+A204S, G182*+D 183*+N195F+M202L+Y203L,R181*+D 184*+N195F+Y200H+M202L+Y203N,R181*+D184*+N195F+Y200Q+M202L+A204S, and R181*+D184*+N195F+M202L+Y203L.6. The variant of claim 1, wherein the variant consists of an alterationselected from the group consisting of: R181*+G182*+Y200H+M202L,R181*+G182*+Y200Q+M202L, R181*+G182*+M202L+Y203N,R181*+G182*+M202L+Y203L, R181*+G182*+M202L+A204S,R181*+G182*+Y200H+M202L+Y203N, R181*+G182*+Y200H+M202L+Y203L,R181*+G182*+Y200H+M202L+A204S, R181*+G182*+Y200Q+M202L+Y203N,R181*+G182*+Y200Q+M202L+Y203L, R181*+G182*+Y200Q+M202L+A204S,R181*+G182*+Y200H+M202L+Y203N+A204S,R181*+G182*+Y200H+M202L+Y203L+A204S,R181*+G182*+Y200Q+M202L+Y203N+A204S,R181*+G182*+Y200Q+M202L+Y203L+A204S, R181*+D183*+Y200H+M202L,R181*+D183*+Y200Q+M202L, R181*+D183*+M202L+Y203N,R181*+D183*+M202L+Y203L, R181*+D183*+M202L+A204S,R181*+D183*+Y200H+M202L+Y203N, R181*+D183*+Y200H+M202L+Y203L,R181*+D183*+Y200H+M202L+A204S, R181*+D183*+Y200Q+M202L+Y203N,R181*+D183*+Y200Q+M202L+Y203L, R181*+D 183*+Y200Q+M202L+A204S,R181*+D183*+Y200H+M202L+Y203N+A204S,R181*+D183*+Y200H+M202L+Y203L+A204S,R181*+D183*+Y200Q+M202L+Y203N+A204S,R181*+D183*+Y200Q+M202L+Y203L+A204S, G182*+G184*+Y200H+M202L,G182*+G184*+Y200Q+M202L, G182*+G184*+M202L+Y203N,G182*+G184*+M202L+Y203L, G182*+G184*+M202L+A204S,G182*+G184*+Y200H+M202L+Y203N, G182*+G184*+Y200H+M202L+Y203L,G182*+G184*+Y200H+M202L+A204S, G182*+G184*+Y200Q+M202L+Y203N,G182*+G184*+Y200Q+M202L+Y203L, G182*+G184*+Y200Q+M202L+A204S,G182*+G184*+Y200H+M202L+Y203N+A204S,G182*+G184*+Y200H+M202L+Y203L+A204S,G182*+G184*+Y200Q+M202L+Y203N+A204S,G182*+G184*+Y200Q+M202L+Y203L+A204S, D183*+G184*+Y200H+M202 L,D183*+G184*+Y200Q+M202 L, D183*+G184*+M202L+Y203N,D183*+G184*+M202L+Y203L, D183*+G184*+M202L+A204S,D183*+G184*+Y200H+M202L+Y203N, D183*+G184*+Y200H+M202L+Y203L,D183*+G184*+Y200H+M202L+A204S, D183*+G184*+Y200Q+M202L+Y203N,D183*+G184*+Y200Q+M202L+Y203L, D183*+G184*+Y200Q+M202L+A204S,D183*+G184*+Y200H+M202L+Y203N+A204S,D183*+G184*+Y200H+M202L+Y203L+A204S,D183*+G184*+Y200Q+M202L+Y203N+A204S, D183*+G184*+Y200Q+M202L+Y203L+A204SN195F+M202L+R181*+G182*, N195F+M202L+R181*+D183*,N195F+M202L+R181*+G184*, N195F+M202L+G182*+D183*,N195F+M202L+D183*+G184*, N195Y+M202L+R181*+G182*,N195Y+M202L+R181*+D183*, N195Y+M202L+R181*+G184*,N195Y+M202L+G182*+D183*, N195Y+M202L+D183*+G184*R181*+G182*+N195F+Y200H+M202L+Y203N,R181*+G182*+N195F+Y200Q+M202L+A204S, R181*+G182*+N195F+M202L+Y203L,G182*+G184*+N195F+Y200H+M202L+Y203N,G182*+G184*+N195F+Y200Q+M202L+A204S, G182*+G184*+N195F+M202L+Y203L,G182*+D183*+N195F+Y200H+M202L+Y203N,G182*+D183*+N195F+Y200Q+M202L+A204S, G182*+D183*+N195F+M202L+Y203L,R181*+D184*+N195F+Y200H+M202L+Y203N,R181*+D184*+N195F+Y200Q+M202L+A204S, and R181*+D184*+N195F+M202L+Y203L.7. The variant of claim 1, the variant comprising an amino acid sequenceof the positions corresponding to positions 198-204 of SEQ ID NO: 1selected from the group consisting of: YDYLLFA, YDWLLYA, YDWLLFA,YDWLLPA, YDYLLIA, YDYLLNA, YDYLLPA, YDYQLYA, YDPLLYA, YDYLLTA, YDYLLWA,YDNLLYA, YDQLLLA, YDQLLYA, YDWLLWA, YDYLLVA, YDQLLPA, YDQLLWA, YDYLLHA,YDYLLLA, YDYLLSA, YDYPLYA, YDYQPAA, YDELLYA, YDKLLPA, YDQLLNA, YDYELYA,YDYHLYA, YDYLLDA, YDYLPRA, YDYQLLA, YDYQLPA, YDYQLQA, YDDLLYA, YDKLLYA,YDWLLHA, YDWLLTA, YDWLPSA, YDWQLYA, YDYGLYA, YDYLLEA, YDYLLQA, YDYPLPA,YDYRLYA, YDIELSA, YDQLLIA, YDQLLSA, YDWLGYA, YDWLLAA, YDWQLHA, YDWWLPA,YDYELLA, YDYLFTA, YDYLLKA, YDYLYSA, YDYQLFA, YDYQYYA, YDYTLYA, YDYYLYA,YDIELWA, YDIELYA, YDNLLNA, YDNLLPA, YDPLLHA, YDQLLVA, YDQLPYA, YDWLLRA,YDWLWYA, YDWWLGA, YDYHLIA, YDYQHYA, YDYQLGA, YDYQLIA, YDYWLPA, YDELLWA,YDHLLNA, YDIELLA, YDIELNA, YDIELRA, YDILLYA, YDKLLWA, YDLPLYA, YDNLLLA,YDPLLAA, YDPLLPA, YDQHLPA, YDQLLEA, YDQLLQA, YDQLNYA, YDQLPFA, YDQLPNA,YDQLPRA, YDTLLLA, YDTLLYA, YDVLLYA, YDWLLKA, YDWLLLA, YDWLLNA, YDWLLQA,YDWLLVA, YDWLPPA, YDWLPTA, YDWPWYA, YDWWLWA, YDYHLFA, YDYHPSA, YDYLLNT,YDYLLYT, YDYLPFA, YDYLPSA, YDYLVSA, YDYLYPA, YDYLYRA, YDYPLFA, YDYPLSA,YDYPLTA, YDYPQYA, YDYQLTA, YDYQLWA, YDYQNYA, YDYQPRA, YDYQSHA, YDYREYA,YDYRLPA, YDYRNSA, YDYRPRA, YDYTQYA, YDYVLYA, YDYWLSA, YDDLLSA, YDELLDA,YDELLPA, YDELLTA, YDEQLEA, YDEQLYA, YDGLPHA, YDIELFA, YDIELKA, YDIELPA,YDKLLNA, YDKPPSA, YDLLLFA, YDLPLLA, YDNLLKA, YDPLKFA, YDPLLEA, YDPLLFA,YDPLLKA, YDPLLWA, YDPPLPA, YDPPLYA, YDPTLPA, YDPTQYA, YDQELPA, YDQLDHA,YDQLEYA, YDQLLDA, YDQLLFA, YDQLLNT, YDQLLYS, YDQLLYT, YDQLPSA, YDQLWYA,YDQQLVA, YDTLLWA, YDTPLFA, YDTPLYA, YDWELYA, YDWHLYA, YDWLHSA, YDWLLEA,YDWLLIA, YDWLLSA, YDWLNYA, YDWLPFA, YDWLPRA, YDWLQPA, YDWLQYA, YDWLWGA,YDWPLHA, YDWQLRA, YDWQLTA, YDWSLPA, YDWSLYA, YDWWLYA, YDYELEA, YDYELNA,YDYGLAA, YDYHEWA, YDYHLPA, YDYHLSA, YDYHQYA, YDYHTSA, YDYLFQA, YDYLHLA,YDYLIEA, YDYLLFT, YDYLLRA, YDYLLYG, YDYLPDA, YDYLPQA, YDYLPWA, YDYLQEA,YDYPGYA, YDYPHSA, YDYPLLA, YDYPLNA, YDYPNYA, YDYPSRA, YDYPWYA, YDYQEYA,YDYQLAA, YDYQLKA, YDYQLPT, YDYQPTA, YDYQPYA, YDYQQYA, YDYQWYA, YDYRLFA,YDYRPSA, YDYRTFA, YDYRTSA, YDYRTYA, YDYSLYA, YDYSVYA, YDYTPRA, YDYWLFA,YDYWLGA, YDYWLWA, YDYWLYA, YDDLLLA, YDDLLNA, YDDLPFA, YDEHLHA, YDELLFA,YDELLSA, YDELQIA, YDEWPYA, YDGLLSA, YDHLLYA, YDIELHA, YDIELKT, YDIELTA,YDIEVSA, YDIPLYA, YDIRGYA, YDIRNYA, YDIRTKA, YDIWLYA, YDKLPHA, YDKLQYA,YDKPLSA, YDLLLVA, YDNHLPA, YDNHLYA, YDNLGYA, YDNLLIA, YDNLLVA, YDNLLWA,YDNLPRA, YDNQLYA, YDNRLYA, YDPHRHA, YDPLHVA, YDPLLDA, YDPLLLA, YDPLQYA,YDPPQFA, YDPQLIA, YDQELYA, YDQLFSA, YDQLKYA, YDQLLAA, YDQLLHA, YDQLNNA,YDQLPAA, YDQLPPA, YDQLQNA, YDQLWGT, YDQLWPA, YDQLYPA, YDQNLYA, YDQPLPA,YDQQLQA, YDQTLYA, YDQWLHA, YDQWLTA, YDRLLPA, YDSELYA, YDTLIRA, YDTLLKA,YDTLLNA, YDTPLNA, YDTPLPA, YDTPQIA, YDTRLYA, YDTSLPA, YDTTLPA, YDTWKYA,YDVLLPA, YDVLNTA, YDWELIA, YDWHLPA, YDWHQYA, YDWHSHA, YDWHTQA, YDWLHHA,YDWLHYA, YDWLNWA, YDWLPAA, YDWLPGA, YDWLPIA, YDWLQVA, YDWLTPA, YDWLTQA,YDWNLSA, YDWNWYA, YDWPGYA, YDWPLIA, YDWPLVA, YDWPLYA, YDWPTYA, YDWQLIA,YDWQLLA, YDWQLNA, YDVWVLDA, YDYDLYA, YDYEKYA, YDYELIA, YDYELPA, YDYELTA,YDYELWA, YDYGWPA, YDYGWYA, YDYHENA, YDYHHEA, YDYHIEA, YDYHLQA, YDYHPRA,YDYHTIA, YDYHTPA, YDYHTYA, YDYLFPA, YDYLHWA, YDYLIRA, YDYLNDA, YDYLNPA,YDYLNQA, YDYLPEA, YDYLPHA, YDYLPIA, YDYLPLA, YDYLPTA, YDYLQNA, YDYLQRA,YDYLRQA, YDYLWGA, YDYLWLA, YDYLWPA, YDYPEPA, YDYPIDA, YDYPIRA, YDYPLAA,YDYPLQA, YDYPPRA, YDYPQHA, YDYPSYA, YDYPTAA, YDYPTDA, YDYQHRA, YDYQHSA,YDYQIYA, YDYQLEA, YDYQLLT, YDYQNPA, YDYQQNA, YDYQQSA, YDYQTVA, YDYQWPA,YDYQYRA, YDYRHTA, YDYRLNA, YDYRQYA, YDYRRSA, YDYRSDA, YDYSGYA, YDYSNYA,YDYSTFA, YDYTEYA, YDYTLSA, YDYTQSA, YDYWLEA, YDYWLGT, YDYWLHA, YDYWLLA,YDYWLTA, YDYWPEA, YDYWPRA, and YDYYLRA.
 8. The variant of claim 1,wherein the deletion a) is selected from the group consisting ofR181*+G182*, R181*+D183*, R181*+G184*, G182*+D183*, G182*+G184*, andD183*+G184*.
 9. The variant of claim 1, wherein the substitution in b)is at two or more of said positions.
 10. The variant of claim 1, whereinthe variant comprises a total of 2-20 alterations.
 11. The variant ofclaim 1, which has at least 91%, at least 92%, at least 93%, at least94%, at least 95% identity, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100%, sequence identity to the amino acidsequence of SEQ ID NO:
 1. 12. The variant of claim 1, which is a variantof a parent alpha-amylase selected from the group consisting of: a. apolypeptide having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 1; and b. a polypeptide having immunologicalcross reactivity with an antibody raised against the mature polypeptideof SEQ ID NO:
 1. 13. The variant of claim 1, wherein the parentalpha-amylase has at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95% identity, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to SEQ ID NO:
 1. 14. Thevariant of claim 1, wherein the parent alpha-amylase comprises orconsists of the mature polypeptide of SEQ ID NO:
 1. 15. The variant ofclaim 1, which has an improved property relative to the parent, whereinthe improved property is selected from the group consisting of catalyticefficiency, catalytic rate, chemical stability, oxidation stability, pHactivity, pH stability, specific activity, stability under storageconditions, substrate binding, substrate cleavage, substratespecificity, substrate stability, surface properties, thermal activity,thermostability, and improved washing performance at low temperature.16. The variant of claim 1, wherein the variant has improvedalpha-amylase activity compared to the alpha-amylase of SEQ ID NO: 1.17. A detergent composition comprising the variant alpha-amylase ofclaim
 1. 18. A dish wash composition comprising the variantalpha-amylase of claim 1.